Antibodies recognizing alpha-synuclein

ABSTRACT

The invention provides monoclonal antibody 5C1 and related antibodies. The 5C1 antibody binds to an epitope within residues 118-126 of α-synuclein. The antibodies of the invention are useful, for example, for treating and/or diagnosing disorders associated with α-synuclein, particularly accumulation of α-synuclein deposits. Such disorders include Lewy body diseases, such as Parkinson&#39;s disease, Diffuse Lewy Body Disease (DLBD), Lewy body variant of Alzheimer&#39;s disease (LBV), Combined Alzheimer&#39;s and Parkinson disease, pure autonomic failure and multiple system atrophy (MSA).

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit under 35 U.S.C. §119(e) ofU.S. Application Nos. 61/711,204 filed Oct. 8, 2012, 61/719,281 filedOct. 26, 2012, 61/840,432 filed Jun. 27, 2013 and 61/872,366 filed Aug.30, 2013, all of which are incorporated by reference in its entirety forall purposes.

SEQUENCE LISTING

A Sequence Listing, comprising SEQ ID NOs: 1-40, is attached andincorporated herein by reference in its entirety. Said listing, in ASCIIformat, was created on ______, is named ______.txt and is ______ bytesin size.

BACKGROUND

Synucleinopathies, also known as Lewy body diseases (LBDs), arecharacterized by degeneration of the dopaminergic system, motoralterations, cognitive impairment, and formation of Lewy bodies (LBs)and/or Lewy neurites. (McKeith et al., Neurology (1996) 47:1113-24).Synucleinopathies include Parkinson's disease (including idiopathicParkinson's disease), Diffuse Lewy Body Disease (DLBD) also known asDementia with Lewy Bodies (DLB), Lewy body variant of Alzheimer'sdisease (LBV), Combined Alzheimer's and Parkinson disease, pureautonomic failure and multiple system atrophy (MSA; e.g.,Olivopontocerebellar Atrophy, Striatonigral Degeneration and Shy-DragerSyndrome). Several nonmotor signs and symptoms are thought to beharbingers for synucleinopathies in the prodromal phase of the diseases(i.e., the presymptomatic, subclinical, preclinical, or premotorperiod). Such early signs include, for example, REM sleep behaviordisorder (RBD), loss of smell and constipation (Mahowald et al.,Neurology (2010) 75:488-489). Lewy body diseases continue to be a commoncause for movement disorders and cognitive deterioration in the agingpopulation (Galasko et al., Arch. Neurol. (1994) 51:888-95).

α-synuclein is part of a large family of proteins including β- andγ-synuclein and synoretin. α-synuclein is expressed in the normal stateassociated with synapses and is believed to play a role in neuralplasticity, learning and memory. Several studies have implicatedα-synuclein with a central role in PD pathogenesis. The protein canaggregate to form insoluble fibrils in pathological conditions. Forexample, synuclein accumulates in LBs (Spillantini et al., Nature (1997)388:839-40; Takeda et al., J. Pathol. (1998) 152:367-72; Wakabayashi etal., Neurosci. Lett. (1997) 239:45-8). Mutations in the α-synuclein geneco-segregate with rare familial forms of parkinsonism (Kruger et al.,Nature Gen. (1998) 18:106-8; Polymeropoulos, et al., Science (1997)276:2045-7). Over expression of α-synuclein in transgenic mice (Masliahet al., Science (2000) 287:1265-9) and Drosophila (Feany et al., Nature(2000) 404:394-8) mimics several pathological aspects of Lewy bodydisease. In addition, it has been suggested that soluble oligomers ofsynuclein may be neurotoxic (Conway et al., Proc Natl Acad Sci USA(2000) 97:571-576; Volles et al., J. Biochem. (2003) 42:7871-7878). Theaccumulation of α-synuclein with similar morphological and neurologicalalterations in species and animal models as diverse as humans, mice, andflies suggests that this molecule contributes to the development of Lewybody disease.

SUMMARY OF THE CLAIMED INVENTION

The invention provides a monoclonal antibody having the three lightchain CDRs as defined by Kabat and three heavy chain CDRs as defined byKabat of monoclonal antibody 5C1, provided that each CDR other thanCDRH2 can have up to four deletions, insertions or substitutions, andCDRH2 can have up to six deletions, insertions or substitutions. 5C1 isa mouse antibody characterized by a heavy chain variable region havingan amino acid sequence comprising SEQ ID NO: 9 and light chain variableregion having an amino acid sequence comprising SEQ ID NO: 24.Optionally, the antibody has the three light chain CDRs as defined byKabat and three heavy chain CDRs as defined by Kabat of monoclonalantibody 5C1. Optionally, the monoclonal antibody is a humanized,chimeric or veneered form of monoclonal antibody 5C1. Optionally, theantibody is an Fab fragment, or single chain Fv. Optionally, theantibody has an isotype of human IgG1. Optionally, the antibody has anisotype of human IgG2 or IgG4 isotype.

The invention provides an antibody comprising a mature heavy chainvariable region having an amino acid sequence at least 90% identical toH4 (SEQ ID NO: 17) and a mature light chain variable region having anamino acid sequence at least 90% identical to L3 (SEQ ID NO: 31),wherein the antibody specifically binds to human alpha synuclein. Somesuch antibodies comprise three Kabat CDRs of SEQ ID NO: 9 and threeKabat CDRs of SEQ ID NO: 24. In some antibodies at least one ofpositions H11, H27, H30, H48, and H73 is occupied by L, Y, T, I, and K,respectively, and at least one of positions L12 and L14 is occupied byS. In some antibodies positions H11, H27, H30, H48, and H73 are occupiedby L, Y, T, I, and K, respectively, and positions L12 and L14 areoccupied by S. In some antibodies, at least one of positions H67, H69,H91, and H94 is occupied by A, L, F, and S, respectively. In someantibodies, positions H67, H69, and H94 are occupied by A, L, and S,respectively. In some antibodies, position H94 is occupied by S. In someantibodies, at least one of positions L2, L45, L49, and L87 is occupiedby V, K, N, and F, respectively. In some antibodies, positions L2, L49,and L87 are occupied by V, N, and F, respectively. Some antibodiescomprise a mature heavy chain variable region having an amino acidsequence at least 95% identical to H4 (SEQ ID NO: 17) and a mature lightchain variable region at least 95% identical to L3 (SEQ ID NO: 31). Insome antibodies any differences in CDRs of the mature heavy chainvariable region and mature light chain variable region from H4 and L3(SEQ ID NOS: 17 and 31, respectively) reside in positions H60-H65.

Some antibodies comprise the mature heavy chain variable region has anamino acid sequence designated H4 (SEQ ID NO: 17) and the mature lightchain variable region has an amino acid sequence designated L3 (SEQ IDNO: 31). Some antibodies comprise the mature heavy chain variable regionhas an amino acid sequence designated H5 (SEQ ID NO: 18) and the maturelight chain variable region has an amino acid sequence designated L3(SEQ ID NO: 31).

In any of the above antibodies, the antibody can have at least onemutation in the constant region. Optionally, the mutation reducescomplement fixation or activation by the constant region. Optionally,the antibody has a mutation at one or more of positions 241, 264, 265,270, 296, 297, 322, 329 and 331 by EU numbering. Optionally, theantibody has alanine at positions 318, 320 and 322.

In any of the above antibodies, the mature heavy chain variable regioncan be fused to a heavy chain constant region and the mature light chainconstant region can be fused to a light chain constant region.

In any of the above antibodies, the heavy chain constant region can be amutant form of natural human constant region which has reduced bindingto an Fcγ receptor relative to the natural human constant region.

In any of the above antibodies, the heavy chain constant region can beof human IgG1 isotype. In some antibodies the allotype is G1m3. In someantibodies, the allotype is G1m1.

The invention also provides a method of humanizing an antibody,comprising determining the sequences of the heavy and light chainvariable regions of a mouse antibody 5C1, synthesizing a nucleic acidencoding a humanized heavy chain comprising CDRs of the mouse antibodyheavy chain and a nucleic acid encoding a humanized light chaincomprising CDRs of the mouse antibody light chain, expressing thenucleic acids in a host cell to produce a humanized antibody.

The invention also provides a method of producing a humanized, chimericor veneered form of antibody 5C1, comprising culturing cells transformedwith nucleic acids encoding the heavy and light chains of the antibody,so that the cell secrete the antibody; and purifying the antibody fromcell culture media.

The invention also provides a method of producing a cell line producinga humanized, chimeric or veneered form of antibody 5C1, comprisingintroducing a vector encoding heavy and light chains of an antibody anda selectable marker into cells; propagating the cells under conditionsto select for cells having increased copy number of the vector;isolating single cells from the selected cells; and banking cells clonedfrom a single cell selected based on yield of antibody. Some suchmethods further comprises propagating the cells under selectiveconditions and screening for cell lines naturally expressing andsecreting at least 100 mg/L/10⁶ cells/24 h.

The invention further provides a pharmaceutical composition comprisingany of the above-mentioned antibodies.

The invention further provides a method of treating or effectingprophylaxis of a Lewy body disease comprising administering an effectiveregime of any of the above-mentioned antibodies and thereby treating oreffecting prophylaxis of the disease.

The invention further provides a method of reducing Lewy body formationin a patient having or at risk of a Lewy body disease, comprisingadministering to the patient an effective amount of any of theabove-mentioned antibodies.

The invention further provides a method of treating a patient having orat risk of a Lewy body disease, comprising administering to the patientan effective regime of any of the above-mentioned antibodies. In somemethods, the disease is Parkinson's disease. In some methods, thedisease is REM sleep behavior disorder (RBD). In some methods, thedisease is Dementia with Lewy Bodies (DLB) or multiple system atrophy(MSA). In some methods, decline of cognitive function in the patient isinhibited. In some methods, neuritic and/or axonal alpha synucleinaggregates are reduced. In some methods, neuritic dystrophy in thepatient is reduced. In some methods, synaptic and/or dendritic densityis preserved. In some methods, the method preserves synaptophysin and/orMAP2 in the patient.

The invention further provides a method of inhibiting synucleinaggregation or reducing Lewy bodies or synuclein aggregates in a patienthaving or at risk of a Lewy body disease, comprising administering tothe patient an effective amount of an antibody as defined by any of theabove-mentioned antibodies. In some such methods, the disease isParkinson's disease. In some methods, decline of cognitive function inthe patient is inhibited. In some methods, neuritic and/or axonal alphasynuclein aggregates are reduced. In some methods, neuritic dystrophy inthe patient is reduced. In some methods, synaptic and/or dendriticdensity is preserved. In some methods, the method preservessynaptophysin and/or MAP2 in the patient.

The invention further provides a method of detecting Lewy bodies in apatient having or at risk of a Lewy body disease, comprisingadministering to the patient an effective amount of any of theabove-mentioned antibodies, wherein the antibody binds to Lewy bodies;and detecting bound antibody in the patient. Optionally, the antibody islabeled.

The invention further provides an isolated nucleic acid, a vector orvectors, and host cells suitable for encoding any of the above-mentionedantibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequences of mouse 5C1 heavy chain maturevariable region. CDR regions according to Kabat definition areunderlined and in bold.

FIG. 2 shows the amino acid sequences of mouse 5C1 light chain maturevariable region. CDR regions according to Kabat definition areunderlined and in bold.

FIG. 3 shows the results of passive immunotherapy with 5C1 on memoryperformance in probe portion of the Morris water maze test.

FIG. 4 shows the results of passive immunotherapy with 5C1 on speed anderrors in the round beam test.

FIG. 5 shows the results of an ELISA assay testing the affinity ofdifferent humanized 5C1 antibodies.

FIG. 6 shows the results of an alanine scanning mutagenesis experimentused to determine the epitope of α-synuclein bound by the 9E4 antibody.The upper portion of the figure shows Western blotts of full lengthα-synuclein (wild-type or individual point mutations of residues118-126, as indicated) stained with 9E4 antibody (left panel) or controlantibody 1H7 (right panel). The lower portion of the figure shows theepitope of α-synuclein bound by the 9E4 antibody.

FIG. 7 shows the results of an alanine scanning mutagenesis experimentused to determine the epitope of α-synuclein bound by the 5C1 antibody.The upper portion of the figure shows Western blotts of full lengthα-synuclein (wild-type or individual point mutations in residues118-126, as indicated) stained with 5C1 antibody (left panel) or controlantibody 1H7 (right panel). The lower portion of the figure shows theepitope of α-synuclein bound by the 5C1 antibody.

FIG. 8 shows the results of an alanine scanning mutagenesis experimentused to determine the epitope of α-synuclein bound by the 5D12 antibody.The upper portion of the figure shows Western blotts of full lengthα-synuclein (wild-type or individual point mutations in residues118-126, as indicated) stained with 5D12 antibody (left panel) orcontrol antibody 1H7 (right panel). The lower portion of the figureshows the epitope of α-synuclein bound by the 5D12 antibody.

FIG. 9 depicts a ball and stick model of the amino acids of α-synucleinproximate to the binding sites of the 9E4, 5C1 and 5D12 antibodies.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is wildtype human α-synuclein.

SEQ ID NO: 2 is the non-amyloid component (NAC) domain of α-synuclein,as reported by Jensen et al. (1995).

SEQ ID NO: 3 is the non-amyloid component (NAC) domain of α-synuclein,as reported by Uéda et al. (1993).

SEQ ID NO: 4 is the 5C1 peptide immunogen amino acid residues 118-129 ofhuman α-synuclein.

VDPDNEAYEGGC

SEQ ID NO: 5 is the nucleotide sequence encoding the murine 5C1 heavychain variable region with sequence encoding signal peptide(underlined).

ATGGAAAGGCACTGGATCTTTCTCTTCCTGTTATCAGTAACTGGAGGTGTCCACTCCCAGGTCCAGCTGCAGCAGTCTGGGGCTGAACTGGCAAAACCTGGGACCTCAGTGCAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAATTACTGGATGAACTGGATAAAAGCGAGGCCTGGACAGGGTCTGGAATGGATTGGGGCTACTAATCCTAACAATGGTTATACTGACTACAATCAGAGGTTCAAGGACAAGGCCATATTAACTGCAGACAAATCCTCCAATACAGCCTACATGCACCTGAGCAGCCTGACATCTGAAGACTCTGCAGTCTATTTCTGTGCAAGTGGGGGGCACTTGGCTTACTGGGGCCAGGGGACTGTGGTCAC TGTCTCTGCA

SEQ ID NO: 6 is the murine 5C1 heavy chain variable region with signalpeptide (underlined).

MERHWIFLFLLSVTGGVHSQVQLQQSGAELAKPGTSVQMSCKASGYTFTNYWMNWIKARPGQGLEWIGATNPNNGYTDYNQRFKDKAILTADKSSNTAYMHLSSLTSEDSAVYFCASGGHLAYWGQGTVVTVSA

SEQ ID NO: 7 is the nucleotide sequence encoding the murine 5C1 lightchain variable region with sequence encoding signal peptide(underlined).

ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTGTGATGACCCAAATTCCACTCTACCTGTCTGTCAGTCCTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTTTCCATAGTAAAGGAAACACCTATTTACATTGGTATCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCAACAGGGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCGGAGTGGAGGCTGAAGATCTGGGAGTTTATTTCTGTTCTCAAAGTGCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAGA

SEQ ID NO: 8 is the murine 5C1 light chain variable region sequence withsignal peptide (underlined)

MKLPVRLLVLMFWIPASSSDVVMTQIPLYLSVSPGDQASISCRSSQSLFHSKGNTYLHWYLQKPGQSPKLLINRVSNRFSGVPDRFSGSGSGTDFTLKISGVEAEDLGVYFCSQSAHVPWTFGGGTKLFIR

SEQ ID NO: 9 is the murine 5C1 mature heavy chain variable region withthe CDRs underlined. The underlined CDRs are as defined by Kabat exceptthe underlined CDRH1 is a composite of Kabat and Chothia definitions.

QVQLQQSGAELAKPGTSVQMSCKASGYTFTNYWMNWIKARPGQGLEWIGATNPNNGYTDYNQRFKDKAILTADKSSNTAYMHLSSLTSEDSAVYFCAS GGHLAYWGQGTVVTVSA

SEQ ID NO: 10 is the sequence of the 5C1 heavy chain CDR1.

NYWMN

SEQ ID NO: 11 is the sequence of the 5C1 heavy chain CDR2.

ATNPNNGYTDYNQRFKD

SEQ ID NO: 12 is the sequence of the 5C1 heavy chain CDR3. GGHLAY

SEQ ID NO: 13 is the human VH Acceptor FR (Acc#AAY42876.1).

QVQLVQSGAEVKKPGSSVKVSCKASGGTFNNYAINWVRQAPGQGLEWMGGIIPIFGTTTYAQKFQGRVTITADESTNTAYMELSSLRSEDTAVYYCAR EGNLNWLDPWGQGTLVTVSS

SEQ ID NO: 14 is the sequence of humanized 5C1H1.

QVQLVQSGAELKKPGSSVKVSCKASGYTFTNYWMNWVRQAPGQGLEWIGATNPNNGYTDYNQRFKDRATLTADKSTNTAYMELSSLRSEDTAVYYCAR GGHLAYWGQGTLVTVSS

SEQ ID NO: 15 is the sequence of humanized 5C1H2.

QVQLVQSGAELKKPGSSVKVSCKASGYTFTNYWMNWVRQAPGQGLEWIGATNPNNGYTDYNQRFKDRVTITADKSTNTAYMELSSLRSEDTAVYYCAR GGHLAYWGQGTLVTVSS

SEQ ID NO: 16 is the sequence of humanized 5C1H3.

QVQLVQSGAELKKPGSSVKVSCKASGYTFTNYWMNWVRQAPGQGLEWIGATNPNNGYTDYNQRFKDRATLTADKSTNTAYMELSSLRSEDTAVYFCAS GGHLAYWGQGTLVTVSS

SEQ ID NO: 17 is the sequence of humanized 5C1H4.

QVQLVQSGAELKKPGSSVKVSCKASGYTFTNYWMNWVRQAPGQGLEWIGATNPNNGYTDYNQRFKDRATLTADKSTNTAYMELSSLRSEDTAVYYCAS GGHLAYWGQGTLVTVSS

SEQ ID NO: 18 is the sequence of humanized 5C1H5.

QVQLVQSGAELKKPGSSVKVSCKASGYTFTNYWMNWVRQAPGQGLEWIGATNPNNGYTDYNQRFKDRVTITADKSTNTAYMELSSLRSEDTAVYYCAS GGHLAYWGQGTLVTVSS

SEQ ID NO: 19 is the nucleic acid sequence encoding humanized 5C1H1 withsequence encoding signal peptide (underlined).

ATGGAGTTCGGCCTGTCCTGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCCAGGTGCAGCTGGTGCAGTCCGGCGCCGAGCTGAAGAAGCCCGGCTCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCACCAACTACTGGATGAACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGCCACCAACCCCAACAACGGCTACACCGACTACAACCAGCGCTTCAAGGACCGCGCCACCCTGACCGCCGACAAGTCCACCAACACCGCCTACATGGAGCTGTCCTCCCTGCGCTCCGAGGACACCGCCGTGTACTACTGCGCCCGCGGCGGCCACCTGGCCTACTGGGGCCAGGGCACCCTGGTGAC CGTGTCCTCC

SEQ ID NO: 20 is the nucleic acid sequence encoding humanized 5C1H2 withsequence encoding signal peptide (underlined).

ATGGAGTTCGGCCTGTCCTGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCCAGGTGCAGCTGGTGCAGTCCGGCGCCGAGCTGAAGAAGCCCGGCTCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCACCAACTACTGGATGAACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGCCACCAACCCCAACAACGGCTACACCGACTACAACCAGCGCTTCAAGGACCGCGTGACCATCACCGCCGACAAGTCCACCAACACCGCCTACATGGAGCTGTCCTCCCTGCGCTCCGAGGACACCGCCGTGTACTACTGCGCCCGCGGCGGCCACCTGGCCTACTGGGGCCAGGGCACCCTGGTGAC CGTGTCCTCC

SEQ ID NO: 21 is the nucleic acid sequence encoding humanized 5C1H3 withsequence encoding signal peptide (underlined).

ATGGAGTTCGGCCTGTCCTGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCCAGGTGCAGCTGGTGCAGTCCGGCGCCGAGCTGAAGAAGCCCGGCTCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCACCAACTACTGGATGAACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGCCACCAACCCCAACAACGGCTACACCGACTACAACCAGCGCTTCAAGGACCGCGCCACCCTGACCGCCGACAAGTCCACCAACACCGCCTACATGGAGCTGTCCTCCCTGCGCTCCGAGGACACCGCCGTGTACTTCTGCGCCTCCGGCGGCCACCTGGCCTACTGGGGCCAGGGCACCCTGGTGAC CGTGTCCTCC

SEQ ID NO: 22 is the nucleic acid sequence encoding humanized 5C1H4 withsequence encoding signal peptide (underlined).

ATGGAGTTCGGCCTGTCCTGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCCAGGTGCAGCTGGTGCAGTCCGGCGCCGAGCTGAAGAAGCCCGGCTCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCACCAACTACTGGATGAACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGCCACCAACCCCAACAACGGCTACACCGACTACAACCAGCGCTTCAAGGACCGCGCCACCCTGACCGCCGACAAGTCCACCAACACCGCCTACATGGAGCTGTCCTCCCTGCGCTCCGAGGACACCGCCGTGTACTACTGCGCCTCCGGCGGCCACCTGGCCTACTGGGGCCAGGGCACCCTGGTGAC CGTGTCCTCC

SEQ ID NO: 23 is the nucleic acid sequence encoding humanized 5C1H5 withsequence encoding signal peptide (underlined).

ATGGAGTTCGGCCTGTCCTGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCCAGGTGCAGCTGGTGCAGTCCGGCGCCGAGCTGAAGAAGCCCGGCTCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCACCAACTACTGGATGAACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGCCACCAACCCCAACAACGGCTACACCGACTACAACCAGCGCTTCAAGGACCGCGTGACCATCACCGCCGACAAGTCCACCAACACCGCCTACATGGAGCTGTCCTCCCTGCGCTCCGAGGACACCGCCGTGTACTACTGCGCCAGCGGCGGCCACCTGGCCTACTGGGGCCAGGGCACCCTGGTGAC CGTGTCCTCC

SEQ ID NO: 24 is the murine 5C1 mature light chain variable regionsequence with the CDRs underlined. The underlined CDRs are as defined byKabat.

DVVMTQIPLYLSVSPGDQASISCRSSQSLFHSKGNTYLHWYLQKPGQSPKLLINRVSNRFSGVPDRFSGSGSGTDFTLKISGVEAEDLGVYFCSQSAH VPWTFGGGTKLEIR

SEQ ID NO: 25 is the sequence of the 5C1 light chain CDR1.

RSSQSLFHSKGNTYLH

SEQ ID NO: 26 is the sequence of the 5C1 light chain CDR2.

RVSNRFS

SEQ ID NO: 27 is the sequence of the 5C1 light chain CDR3.

SQSAHVPWT

SEQ ID NO: 28 is the human VL Acceptor FR (Acc#CAB51293.1).

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQ TPPTFGGGTKVEIK

SEQ ID NO: 29 is the sequence of humanized 5C1L1.

DVVMTQSPLSLSVSPGEPASISCRSSQSLFHSKGNTYLHWYLQKPGQSPKLLINRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSAH VPWTFGGGTKVEIK

SEQ ID NO: 30 is the sequence of humanized 5C1L2.

DIVMTQSPLSLSVSPGEPASISCRSSQSLFHSKGNTYLHWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSAH VPWTFGGGTKVEIK

SEQ ID NO: 31 is the sequence of humanized 5C1L3.

DVVMTQSPLSLSVSPGEPASISCRSSQSLFHSKGNTYLHWYLQKPGQSPQLLINRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSAH VPWTFGGGTKVEIK

SEQ ID NO: 32 is the sequence of humanized 5C1L4.

DIVMTQSPLSLSVSPGEPASISCRSSQSLFHSKGNTYLHWYLQKPGQSPQLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSAH VPWTFGGGTKVEIK

SEQ ID NO: 33 is the nucleic acid sequence encoding humanized 5C1L1 withsequence encoding signal peptide (underlined).

ATGGACATGCGCGTGCCCGCCCAGCTGCTGGGCCTGCTGATGCTGTGGGTGTCCGGCTCCTCCGGCGACGTGGTGATGACCCAGTCCCCCCTGTCCCTGTCCGTGTCCCCCGGCGAGCCCGCCTCCATCTCCTGCCGCTCCTCCCAGTCCCTGTTCCACTCCAAGGGCAACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGTCCCCCAAGCTGCTGATCAACCGCGTGTCCAACCGCTTCTCCGGCGTGCCCGACCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGAAGATCTCCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTTCTGCTCCCAGTCCGCCCACGTGCCCTGGACCTTCGGCGGCGGCACCAAGGT GGAGATCAAG

SEQ ID NO: 34 is the nucleic acid sequence encoding humanized 5C1L2 withsequence encoding signal peptide (underlined).

ATGGACATGCGCGTGCCCGCCCAGCTGCTGGGCCTGCTGATGCTGTGGGTGTCCGGCTCCTCCGGCGACATCGTGATGACCCAGTCCCCCCTGTCCCTGTCCGTGTCCCCCGGCGAGCCCGCCTCCATCTCCTGCCGCTCCTCCCAGTCCCTGTTCCACTCCAAGGGCAACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGTCCCCCAAGCTGCTGATCTACCGCGTGTCCAACCGCTTCTCCGGCGTGCCCGACCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGAAGATCTCCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCTCCCAGTCCGCCCACGTGCCCTGGACCTTCGGCGGCGGCACCAAGGT GGAGATCAAG

SEQ ID NO: 35 is the nucleic acid sequence encoding humanized 5C1L3 withsequence encoding signal peptide (underlined).

ATGGACATGCGCGTGCCCGCCCAGCTGCTGGGCCTGCTGATGCTGTGGGTGTCCGGCTCCTCCGGCGACGTGGTGATGACCCAGTCCCCCCTGTCCCTGTCCGTGTCCCCCGGCGAGCCCGCCTCCATCTCCTGCCGCTCCTCCCAGTCCCTGTTCCACTCCAAGGGCAACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGTCCCCCCAGCTGCTGATCAACCGCGTGTCCAACCGCTTCTCCGGCGTGCCCGACCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGAAGATCTCCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTTCTGCTCCCAGTCCGCCCACGTGCCCTGGACCTTCGGCGGCGGCACCAAGGT GGAGATCAAG

SEQ ID NO: 36 is the nucleic acid sequence encoding humanized 5C1L4 withsequence encoding signal peptide (underlined).

ATGGACATGCGCGTGCCCGCCCAGCTGCTGGGCCTGCTGATGCTGTGGGTGTCCGGCTCCTCCGGCGACATCGTGATGACCCAGTCCCCCCTGTCCCTGTCCGTGTCCCCCGGCGAGCCCGCCTCCATCTCCTGCCGCTCCTCCCAGTCCCTGTTCCACTCCAAGGGCAACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGTCCCCCCAGCTGCTGATCTACCGCGTGTCCAACCGCTTCTCCGGCGTGCCCGACCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGAAGATCTCCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCTCCCAGTCCGCCCACGTGCCCTGGACCTTCGGCGGCGGCACCAAGGT GGAGATCAAG

SEQ ID NO: 37 is the nucleic acid sequence encoding an exemplary humanIgG1 constant region.

GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGTCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACGCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA

SEQ ID NO: 38 is the amino acid sequence of an exemplary human IgG1constant region.

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNVKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 39 is the nucleic acid sequence encoding an exemplary humankappa light chain constant region.

ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

SEQ ID NO: 40 is the amino acid sequence of an exemplary human kappalight chain constant region.

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC

DEFINITIONS

The basic antibody structural unit is a tetramer of subunits. Eachtetramer includes two identical pairs of polypeptide chains, each pairhaving one “light” chain (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. When initially expressed, this variable region istypically linked to a cleavable signal peptide. The variable regionwithout the signal peptide is sometimes referred to as a mature variableregion. Thus, for example, a light chain mature variable region means alight chain variable region without the light chain signal peptide. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. A constant region caninclude any or all of a CH1 region, hinge region, CH2 region, and CH3region.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 or more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989),Ch. 7) (incorporated by reference in its entirety for all purposes).

The mature variable regions of each light/heavy chain pair form theantibody binding site. Thus, an intact antibody has two binding sites.Except for bifunctional or bispecific antibodies, the two binding sitesare the same. The chains all exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise theregions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of aminoacids to each region is in accordance with the definitions of Kabat,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol.196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). Kabatalso provides a widely used numbering convention (Kabat numbering) inwhich corresponding residues between different heavy chains or betweendifferent light chains are assigned the same number (e.g., H83 meansposition 83 by Kabat numbering in the mature heavy chain variableregion; likewise position L36 means position 36 by Kabat numbering inthe mature light chain variable region). Kabat numbering is usedthroughout in referring to positions in the variable region of anantibody unless explicitly stated otherwise.

The term “antibody” includes intact antibodies and binding fragmentsthereof. Typically, fragments compete with the intact antibody fromwhich they were derived for specific binding to the target. Fragmentsinclude separate heavy chains, separate light chains, Fab, Fab′,F(ab′)2, F(ab)c, Fv, single chain antibodies, and single domainantibodies. Single (variable) domain antibodies include VH regionsseparated from their VL partners (or vice versa) in conventionalantibodies (Ward et al., 1989, Nature 341: 544-546), as well as VHregions (sometimes known as VHH) from species such as Camelidae orcartilaginous fish (e.g., a nurse shark) in which VH regions are notassociated with VL regions (see, e.g., WO 9404678). Single domainantibodies in which one chain is separated from its natural partners aresometimes known as Dabs and single domain antibodies from Caemelidae orcartilaginous fish are sometimes known as nanobodies. Constant regionsor parts of constant regions may or may not be present in single domainantibodies. For example, natural single variable region antibodies fromCamelidae include a VHH variable region, and CH2 and CH3 constantregions. Single domain antibodies, such as nanobodies, can be subject tohumanization by analogous approaches to conventional antibodies. Dabsantibodies are usually obtained from antibodies of human origin.Fragments can be produced by recombinant DNA techniques, or by enzymaticor chemical separation of intact immunoglobulins.

The term “antibody” also includes a bispecific antibody and/or ahumanized antibody. A bispecific or bifunctional antibody is anartificial hybrid antibody having two different heavy/light chain pairsand two different binding sites (see, e.g., Songsivilai and Lachmann,Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol.148:1547-53 (1992)). In some bispecific antibodies, the two differentheavy/light chain pairs include a humanized 5C1 heavy chain/light chainpair and a heavy chain/light chain pair specific for a different epitopeon alpha synuclein than that bound by 5C1.

In some bispecific antibodies, one heavy chain light chain pair is ahumanized 5C1 antibody as further disclosed below and the heavy lightchain pair is from an antibody that binds to a receptor expressed on theblood brain barrier, such as an insulin receptor, an insulin-like growthfactor (IGF) receptor, a leptin receptor, or a lipoprotein receptor, ora transferrin receptor (Friden et al., PNAS 88:4771-4775, 1991; Fridenet al., Science 259:373-377, 1993). Such a bispecific antibody can betransferred cross the blood brain barrier by receptor-mediatedtranscytosis. Brain uptake of the bispecific antibody can be furtherenhanced by engineering the bi-specific antibody to reduce its affinityto the blood brain barrier receptor. Reduced affinity for the receptorresulted in a broader distribution in the brain (see, e.g., Atwal. etal. Sci. Trans. Med. 3, 84ra43, 2011; Yu et al. Sci. Trans. Med. 3,84ra44, 2011).

Exemplary bispecific antibodies can also be (1) a dual-variable-domainantibody (DVD-Ig), where each light chain and heavy chain contains twovariable domains in tandem through a short peptide linkage (Wu et al.,Generation and Characterization of a Dual Variable Domain Immunoglobulin(DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg(2010)); (2) a Tandab, which is a fusion of two single chain diabodiesresulting in a tetravalent bispecific antibody that has two bindingsites for each of the target antigens; (3) a flexibody, which is acombination of scFvs with a diabody resulting in a multivalent molecule;(4) a so called “dock and lock” molecule, based on the “dimerization anddocking domain” in Protein Kinase A, which, when applied to Fabs, canyield a trivalent bispecific binding protein consisting of two identicalFab fragments linked to a different Fab fragment; (5) a so-calledScorpion molecule, comprising, e.g., two scFvs fused to both termini ofa human Fc-region. Examples of platforms useful for preparing bispecificantibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2(F-star), Fc-engineered IgG1 (Xencor) or DuoBody (based on Fab armexchange, Genmab).

An “antigen” is an entity to which an antibody specifically binds.

The term “epitope” refers to a site on an antigen to which an antibodybinds. For protein antigens, an epitope can be formed from contiguousamino acids or noncontiguous amino acids juxtaposed by tertiary foldingof one or more proteins. Epitopes formed from contiguous amino acids aretypically retained on exposure to denaturing solvents, whereas epitopesformed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 2, 3, andmore usually, at least 5 or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, X-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). An epitope caninclude a C-terminal residue or an N-terminal residue. An epitope canalso include, but need not include, the free amino group of apolypeptide or the free carboxyl group of a polypeptide. Thus, anepitope can include a C-terminal or an N-terminal residue, but notnecessarily include the free carboxyl group or the free amino group,respectively. Antibody binding specificity is sometimes defined by arange of amino acids. If an antibody is said to bind to an epitopewithin amino acids 118-126 of SEQ ID NO:1, for example, what is meant isthat the epitope is within the recited range of amino acids includingthose defining the outer-limits of the range. It does not necessarilymean that every amino acid within the range constitutes part of theepitope. Thus, for example, an epitope within amino acids 118-126 of SEQID NO:1 may consist of amino acids 118-124, 119-125, 120-126, 120-124,or 120-122, among other segments of SEQ ID NO:1.

Antibodies that recognize the same or overlapping epitopes can beidentified in a simple immunoassay showing the ability of one antibodyto compete with the binding of another antibody to a target antigen. Theepitope of an antibody can also be defined by X-ray crystallography ofthe antibody bound to its antigen to identify contact residues (theepitope being defined by the residues making contact). Alternatively,two antibodies have the same epitope if all amino acid mutations in theantigen that reduce or eliminate binding of one antibody reduce oreliminate binding of the other. Two antibodies have overlapping epitopesif some amino acid mutations that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other.

Competition between antibodies is determined by an assay in which anantibody under test inhibits specific binding of a reference antibody toa common antigen. See, e.g., Junghans et al. (1990), Cancer Res.50:1495. A test antibody competes with a reference antibody if an excessof a test antibody (e.g., at least 2×, 5×, 10×, 20× or 100×) inhibitsbinding of the reference antibody by at least 50%, 75%, 90%, 95%, 98%,or 99% as measured in a competitive binding assay. Antibodies identifiedby competition assay (competing antibodies) include antibodies bindingto the same epitope as the reference antibody and antibodies binding toan adjacent epitope sufficiently proximal to the epitope bound by thereference antibody for steric hindrance to occur.

Antibodies of the invention typically bind to their designated targetwith an affinity constant of at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M−1.Such binding is specific binding in that it is detectably higher inmagnitude and distinguishable from non-specific binding occurring to atleast one unrelated target. Specific binding can be the result offormation of bonds between particular functional groups or particularspatial fit (e.g., lock and key type) whereas nonspecific binding isusually the result of van der Waals forces. Specific binding does nothowever necessarily imply that a monoclonal antibody binds one and onlyone target.

When comparing antibody sequences, percentage sequence identities aredetermined with antibody sequences maximally aligned by the Kabatnumbering convention. After alignment, if a subject antibody region(e.g., the entire mature variable region of a heavy or light chain) isbeing compared with the same region of a reference antibody, thepercentage sequence identity between the subject and reference antibodyregions is the number of positions occupied by the same amino acid inboth the subject and reference antibody region divided by the totalnumber of aligned positions of the two regions, with gaps not counted,multiplied by 100 to convert to percentage.

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic side chains): met, ala, val, leu, ile; Group II (neutralhydrophilic side chains): cys, ser, thr; Group III (acidic side chains):asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V(residues influencing chain orientation): gly, pro; and Group VI(aromatic side chains): trp, tyr, phe. Conservative substitutionsinvolve substitutions between amino acids in the same class.Non-conservative substitutions constitute exchanging a member of one ofthese classes for a member of another.

Monoclonal antibodies are typically provided in isolated form. Thismeans that the antibody is typically at least 50% w/w pure ofinterfering proteins and other contaminants arising from its productionor purification, but does not exclude the possibility that the agent iscombined with an excess of pharmaceutically-acceptable carrier(s) orother vehicle intended to facilitate its use. Sometimes monoclonalantibodies are at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%w/w pure of aggregates or fragments of such monoclonal antibodies or ofother proteins and contaminants. Some such monoclonal antibodies mayinclude aggregates or fragments but are at least 99% w/w pure of otherproteins and contaminants.

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises antibody may contain the antibody alone or incombination with other ingredients.

Designation of a range of values includes all integers within ordefining the range, and all subranges defined by integers within therange.

Unless otherwise apparent from the context, the term “about” encompassesvalues within the margin of error of measurement (SEM) of a statedvalue.

Statistical significance means p≦0.05.

A “patient” includes a human or other mammalian subject that receiveseither prophylactic or therapeutic treatment.

An individual is at increased risk of a disease if the subject has atleast one known risk-factor (e.g., genetic, biochemical, family history,situational exposure) placing individuals with that risk factor at astatistically significant greater risk of developing the disease thanindividuals without the risk factor.

The term “symptom” refers to a subjective evidence of a disease, such asaltered gait, as perceived by the patient. A “sign” refers to objectiveevidence of a disease as observed by a physician.

“Cognitive function” refers to mental processes such as any or all ofattention, memory, producing and understanding language, solvingproblems, and taking an interest in one's surroundings and self-care.

“Enhanced cognitive function” or “improved cognitive function” refers toimprovement relative to a baseline, for example, diagnosis or initiationof treatment. “Decline of cognitive function” refers to a decrease infunction relative to such a base line.

In animal model systems such as rat or mouse, cognitive function may bemeasured methods including using a maze in which subjects use spatialinformation (e.g., Morris water maze, Barnes circular maze, elevatedradial arm maze, T maze and others), fear conditioning, activeavoidance, illuminated open-field, dark activity meter, elevatedplus-maze, two-compartment exploratory test or forced swimming test.

In humans, cognitive function can be measured by one or more of severalstandardized tests. Examples of a test or assay for cognitive functionwere described (Ruoppila and Suutama, Scand. J. Soc. Med. Suppl.53,44-65, 1997) and include standardized psychometric tests (e. g.Wechsler Memory Scale, the Wechsler Adult Intelligence Scale, Raven'sStandard Progressive Matrices, Schaie-Thurstone Adult Mental AbilitiesTest), neuropsychological tests (e. g. Luria-Nebraska), metacognitiveself-evaluations (e. g. Metamemory Questionnaire), visual-spatialscreening tests (e.g. Poppelreuter's Figures, Clock Recognition,Honeycomb Drawing and Cancellation), cognitive screening tests (e. g.Folstein's Mini Mental State Test) and reaction time tests. Otherstandard tests for cognitive performance include the Alzheimer's DiseaseAssessment Scale-cognitive subscale (ADAS-cog); the clinical globalimpression of change scale (CIBIC-plus scale); the Alzheimer's DiseaseCooperative Study Activities of Daily Living Scale (ADCS-ADL); the MiniMental State Exam (MMSE); the Neuropsychiatric Inventory (NPI); theClinical Dementia Rating Scale (CDR); the Cambridge NeuropsychologicalTest Automated Battery (CANTAB) or the Sandoz ClinicalAssessment-Geriatric (SCAG), Stroop Test, Trail Making, Wechsler DigitSpan, and the CogState computerized cognitive test. In addition,cognitive function may be measured using imaging techniques such asPositron Emission Tomography (PET), functional magnetic resonanceimaging (fMRI), Single Photon Emission Computed Tomography (SPECT), orany other imaging technique that allows one to measure brain function.

DETAILED DESCRIPTION OF THE INVENTION I. General

The invention provides monoclonal antibody 5C1 and related antibodies,such as antibodies that bind to the same epitope on α-synuclein (i.e.,an epitope 118-126 of α-synuclein). The antibodies of the invention areuseful, for example, for treating disorders associated with α-synucleinaccumulation, particularly accumulation in Lewy bodies. Such disordersinclude Lewy Body Diseases, such as Parkinson's disease, Diffuse LewyBody Disease (DLBD), Lewy body variant of Alzheimer's disease (LBV),Combined Alzheimer's and Parkinson disease, pure autonomic failure andmultiple system atrophy (MSA). The antibodies are also useful fordiagnoses of a Lewy Body Diseases.

II. Target Molecules

Natural human wildtype α-synuclein is a peptide of 140 amino acidshaving the following amino acid sequence:

(SEQ ID NO: 1) MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA(Uéda et al., Proc. Natl. Acad. Sci. USA, 90:11282-6, 1993; GenBankaccession number: P37840). The protein has three recognized domains, aKTKE repeat domain covering amino acids 1-61, a NAC (Non-amyloidcomponent) domain running from about amino acids 60-95, and a C-terminalacidic domain running from about amino acid 98 to 140. Jensen et al.(1995) reported that NAC has the amino acid sequence:

(SEQ ID NO: 2) EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFV(Jensen et al., Biochem. J. 310.1: 91-94; GenBank accession numberS56746). However, Ueda et al. (1993) reported that NAC has the aminoacid sequence:

(SEQ ID NO: 3) KEQVTNVGGAVVTGVTAVAQKTVEGAGS(Uéda et al., Proc. Natl. Acad. Sci. USA, 90:11282-6).

Unless otherwise apparent from the context, reference to α-synuclein orits fragments includes the natural human wildtype amino acid sequencesindicated above, and human allelic variants thereof, particularly thoseassociated with Lewy body disease (e.g., E46K, A30P and A53T, with thefirst letter indicates the amino acid in SEQ ID NO:1, the number is thecodon position in SEQ ID NO:1, and the second letter is the amino acidin the allelic variant). Such variants can optionally be presentindividually or in any combination. The induced mutations E83Q, A90V,A76T, which enhance alpha synuclein aggregation, can also be presentindividually or in combination with each other and/or human allelicvariants E46K, A30P and A53T.

III. Lewy Body Diseases

Lewy Body Diseases (LBD) are characterized by degeneration of thedopaminergic system, motor alterations, cognitive impairment, andformation of Lewy bodies (LBs) (McKeith et al., Neurology (1996)47:1113-24). Lewy Bodies are spherical protein deposits found in nervecells. Their presence in the brain disrupts the brain's normal functioninterrupting the action of chemical messengers including acetylcholineand dopamine. Lewy Body diseases include Parkinson's disease (includingidiopathic Parkinson's disease), Diffuse Lewy Body Disease (DLBD) alsoknown as Dementia with Lewy Bodies (DLB), Lewy body variant ofAlzheimer's disease (LBV), Combined Alzheimer's and Parkinson diseaseand as multiple system atrophy (MSA; e.g., Olivopontocerebellar Atrophy,Striatonigral Degeneration and Shy-Drager Syndrome). DLBD sharessymptoms of both Alzheimer's and Parkinson's disease. DLBD differs fromParkinson's disease mainly in the location of Lewy Bodies. In DLBD, LewyBodies form mainly in the cortex. In Parkinson's disease, they formmainly in the substantia nigra. Other Lewy Body diseases include PureAutonomic Failure, Lewy body dysphagia, Incidental LBD, and InheritedLBD (e.g., mutations of the α-synuclein gene, PARK3 and PARK4).

IV. Antibodies A. Binding Specificity and Functional Properties

5C1 is an exemplary antibody of the invention, whose heavy and lightchain mature variable regions are designated SEQ ID NO: 9 and SEQ ID NO:24, respectively. The invention also provides antibodies competing with5C1 for binding to α-synuclein, or which bind to the same or overlappingepitope as 5C1, and have similar functional properties, such as reducingneuronal aggregates of α-synuclein, improving cognitive function, and/orpreserving synaptic density and/or dentritic density.

Other antibodies having such binding specificity can be produced byimmunizing mice with α-synuclein or a fragment thereof (e.g., a fragmentincluding amino acid residues 118-126, or a portion thereof), andscreening the resulting antibodies for binding to α-synuclein,optionally in competition with 5C1. Use of a fragment is preferred forgenerating an antibody having the same epitope as 5C1. Antibodies canalso be screened for their effect in: (1) α-synuclein transgenic rodentmodels subjected to behavioral assays, such as the Morris Water Maze(MWM) test or horizontal beam test, and/or immunological assays for thedetection of α-synuclein, α-synuclein aggregation, synaptophysin, MAP2,and/or PSD95 in brain tissue; (2) rodent or other non-human animalmodels for a disease characterized by α-synuclein accumulation, usingbehavioral assays such as the Morris Water Maze (MWM) test or horizontalbeam test and/or immunological assays for the detection of α-synuclein,α-synuclein aggregation, synaptophysin, MAP2, and/or PSD95 in braintissue; and/or (3) humans with a condition associated with α-synucleinaccumulation, using appropriate behavioral assays. Alternatively, or inaddition to any of the foregoing approaches, antibodies can be screenedagainst mutagenized forms of α-synuclein to identify an antibody showingthe same or similar binding profile as 5C1 to a collection of mutationalchanges. The mutations can be systematic substitution with alanine (orserine if an alanine is present already) one residue at a time, or morebroadly spaced intervals, throughout α-synuclein or through a sectionthereof in which the epitope is known to reside (e.g., residues118-126).

FIGS. 6-8 and Example 6 characterize the epitope of 5C1 in comparisonwith two other antibodies binding within residues 118-126, namely 9E4and 5D12. Alanine mutagenesis tests the effect of mutating individualamino acids, one at a time, in the 118-126 of alpha synuclein. Theprofile of relative changes of binding affinity (in other words,contribution to binding) caused by mutation of different amino acidswithin residues 118-126 characterizes the epitope. For 5C1, mutagenesisof any of residues 120-122 has the greatest reduction of binding.Mutagenesis of residue 123 or 124 results in a significant reduction ofbinding, but not as much as at any of positions 120-122. Mutagenesis ofresidue 118, 119, 125 or residue 126 results in still less loss ofbinding affinity, essentially unchanged. For simplicity, the effects ofmutagenesis can be approximately subdivided into three categories:essentially complete reduction of binding for residues 120-122(indistinguishable from negative control), essentially no reduction ofbinding for residues 118, 119, 125 and 126 (indistinguishable frompositive control) and intermediate reduction of binding affinity forresidues 123 and 124. The epitope of 5C1 can thus be approximatelycharacterized as a linear epitope consisting of or consistingessentially of residues 120-124 of SEQ ID NO:1, with residues 120-122making the greatest contribution to binding. Antibodies having the 5C1epitope as characterized by any of the descriptions in this paragraphare provided. Some antibodies are characterized by an epitope consistingessentially of residues 120-122 and excluding residues 119-120 meaningthat residues 120-122 each contribute more to binding than any otherresidue and residues 119 and 120 make no detectable contribution tobinding (e.g., by the alanine scanning method of the example). Residues123 and 124 may or may not make a minor contribution to binding in suchantibodies.

For 5D12, the epitope is characterized by alanine mutagenesis asfollows. Mutagenesis of any of residues 120-122 has the greatestreduction of binding. Mutagenesis of residue 118, 199, 123 or 124results in significant reduction of binding, but not as much as at anyof positions 120-122. Mutagenesis of residue 125 or residue 126 resultsin still less loss of binding affinity, essentially unchanged. Forsimplicity, the effects of mutagenesis can be approximately subdividedinto three categories: essentially complete reduction of binding formutation of residues 120-122 (indistinguishable from negative control),essentially no reduction of binding for residues 125 and 126(indistinguishable from positive control) and intermediate reduction ofbinding affinity for residues 118, 119, 123 and 124. The epitope of 5D12can thus be approximately characterized as a linear epitope consistingof or consisting essentially of residues 118-124 of SEQ ID NO:1, withresidues 120-122 making the greatest contribution to binding. Otherantibodies having the epitope of 5D12 as characterized by any of thedescriptions in this paragraph or Example 6 are also provided.

Likewise, the 9E4 epitope can be characterized by mutation of residues122 and 125 each showing greater reduction of binding than any ofresidues 118-121, 123, 124 or 126. For simplicity the effects ofmutagenesis can be approximately divided into two categories:essentially complete reduction of binding for residues 122 and 125 andessentially no reduction in binding for residues 118-121, 123, 124 and126. The 9E4 epitope can thus be approximately characterized as aconformational epitope in which residues 122 and 125 provide the contactpoints (or greatest contribution to binding) with 9E4. Other antibodieshaving the 9E4 epitope as characterized by any of the descriptions inthis paragraph or Example 6 are provided. Optionally, the antibody isnot 9E4 or other antibody having the same CDRs as 9E4 nor an antibodyhaving at least five Kabat CDRs with at least 85% sequence identity tothe corresponding CDRs of 9E4.

Antibodies having the binding specificity of a selected murine antibody(e.g., 5C1) can also be produced using a variant of the phage displaymethod. See Winter, WO 92/20791. This method is particularly suitablefor producing human antibodies. In this method, either the heavy orlight chain variable region of the selected murine antibody is used as astarting material. If, for example, a light chain variable region isselected as the starting material, a phage library is constructed inwhich members display the same light chain variable region (i.e., themurine starting material) and a different heavy chain variable region.The heavy chain variable regions can for example be obtained from alibrary of rearranged human heavy chain variable regions. A phageshowing strong specific binding for α-synuclein (e.g., at least 10⁸ M⁻¹,and preferably at least 10⁹ M⁻¹) is selected. The heavy chain variableregion from this phage then serves as a starting material forconstructing a further phage library. In this library, each phagedisplays the same heavy chain variable region (i.e., the regionidentified from the first display library) and a different light chainvariable region. The light chain variable regions can be obtained forexample from a library of rearranged human variable light chain regions.Again, phage showing strong specific binding for sa-synuclein areselected. The resulting antibodies usually have the same or similarepitope specificity as the murine starting material.

Other antibodies can be obtained by mutagenesis of cDNA encoding theheavy and light chains of an exemplary antibody, such as 5C1.Accordingly, monoclonal antibodies that are at least 70%, 80%, 90%, 95%,96%, 97%, 98% or 99% identical to 5C1 in amino acid sequence of themature heavy and/or light chain variable regions and maintain itsfunctional properties, and/or which differ from the respective antibodyby a small number of functionally inconsequential amino acidsubstitutions (e.g., conservative substitutions), deletions, orinsertions are also included in the invention.

The invention also includes monoclonal antibodies having some or all(e.g., 3, 4, 5, and preferably 6) CDRs entirely or substantially from5C1. Such antibodies can include a heavy chain variable region that hasat least two, and usually all three, CDRs entirely or substantially fromthe heavy chain variable region of 5C1 and/or a light chain variableregion having at least two, and usually all three, CDRs entirely orsubstantially from the light chain variable region of 5C1. Preferredantibodies include both heavy and light chains. A CDR is substantiallyfrom a corresponding 5C1 CDR when it contains no more than 4, 3, 2 or 1substitutions, insertions or deletions, except that CDRH2 (when definedby Kabat) can have no more than 6, 5, 4, 3, 2, or 1 substitutions,insertion or deletions. Such antibodies preferably have at least 70%,80%, 90%, 95%, 96%, 97%, 98% or 99% identity to 5C1 in the amino acidsequence of the mature heavy and/or light chain variable regions andmaintain its functional properties, and/or differ from 5C1 by a smallnumber of functionally inconsequential amino acid substitutions (e.g.,conservative substitutions), deletions, or insertions.

Preferred antibodies show similar functional activity to 5C1, e.g.,reducing neuritic and/or axonal α-synuclein aggregates, reducingneuritic dystrophy, improving cognitive function, reversing, treating orinhibiting cognitive decline, and/or preserving or increasing synapticdensity and/or dentritic density.

B. Chimeric and Veneered Antibodies

The invention further provides chimeric and veneered forms of non-humanantibodies, particularly 5C1.

A chimeric antibody is an antibody in which the mature variable regionsof light and heavy chains of a non-human antibody (e.g., a mouse) arecombined with human light and heavy chain constant regions. Typically,the light and heavy chain constant regions are of human origin, but theconstant regions can originate from a different non-human species asneeded (e.g., to facilitate testing of the non-human antibody in anappropriate animal model). Such antibodies substantially or entirelyretain the binding specificity of the mouse antibody, and can be abouttwo-thirds human sequence contributed by the human constant regions.

A veneered antibody is a type of humanized antibody that retains someand usually all of the CDRs and some of the non-human variable regionframework residues of a non-human antibody but replaces other variableregion framework residues that may contribute to B- or T-cell epitopes,for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991) withresidues from the corresponding positions of a human antibody sequence.The result is an antibody in which the CDRs are entirely orsubstantially from a non-human antibody and the variable regionframeworks of the non-human antibody are made more human-like by thesubstitutions.

C. Humanized Antibodies

Humanized 5C1 antibodies specifically bind to human α-synuclein. Theaffinity of some humanized antibodies (i.e., Ka) is can be, for example,within a factor of five or two of that of the murine 5C1 antibody. Somehumanized antibodies have an affinity that is the same, withinexperimental error, as murine 5C1. Some humanized antibodies have anaffinity greater than that of murine 5C1. Preferred humanized antibodiesbind to the same epitope and/or compete with murine 5C1 for binding tohuman α-synuclein.

A humanized antibody is a genetically engineered antibody in which theCDRs from a non-human “donor” antibody (e.g., murine 5C1) are graftedinto human “acceptor” antibody sequences (see, e.g., Queen, U.S. Pat.Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539, Carter,U.S. Pat. No. 6,407,213, Adair, U.S. Pat. No. 5,859,205 6,881,557,Foote, U.S. Pat. No. 6,881,557). The acceptor antibody sequences can be,for example, a mature human antibody sequence, a composite of suchsequences, a consensus sequence of human antibody sequences, or agermline region sequence. Thus, a humanized 5C1 antibody is an antibodyhaving some or all CDRs entirely or substantially from murine 5C1 andvariable region framework sequences and constant regions, if present,entirely or substantially from human antibody sequences. Similarly ahumanized heavy chain has at least two and usually all three CDRsentirely or substantially from a donor antibody heavy chain, and a heavychain variable region framework sequence and heavy chain constantregion, if present, substantially from human heavy chain variable regionframework and constant region sequences. Similarly a humanized lightchain has at least two and usually all three CDRs entirely orsubstantially from a donor antibody light chain, and a light chainvariable region framework sequence and light chain constant region, ifpresent, substantially from human light chain variable region frameworkand constant region sequences. Other than nanobodies and dAbs, ahumanized antibody comprises a humanized heavy chain and a humanizedlight chain. Preferably at least 85%, 90%, 95% or 100% of correspondingresidues (as defined by Kabat) are identical between the respectiveCDRs. The variable region framework sequences of an antibody chain orthe constant region of an antibody chain are substantially from a humanvariable region framework sequence or human constant region,respectively, when at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%of corresponding residues defined by Kabat are identical.

Although humanized antibodies often incorporate all six CDRs (preferablyas defined by Kabat) from a mouse antibody, they can also be made withless than all CDRs (at least 3, 4, or 5) CDRs from a mouse antibody(e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al.,Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol.Immunol. 36:1079-1091, 1999; Tamura et al, Journal of Immunology,164:1432-1441, 2000).

In some antibodies only part of the CDRs, namely the subset of CDRresidues required for binding, termed the specificity determiningresidues (SDRs) (Kashmiri et al., Methods (2005) 36 (1):25-34), areneeded to retain binding in a humanized antibody. CDR residues notcontacting antigen and not in the SDRs can be identified based onprevious studies (for example one or more or all of residues H60-H65 inCDR H2 are sometimes not required), from regions of Kabat CDRs lyingoutside Chothia hypervariable loops (Chothia, J. Mol. Biol. 196:901,1987), by molecular modeling and/or empirically, or as described inGonzales et al., Mol. Immunol. 41: 863, 2004. In such humanizedantibodies, at positions in which one or more donor CDR residues isabsent or in which an entire donor CDR is omitted, the amino acidoccupying the position can be an amino acid occupying the correspondingposition (by Kabat numbering) in the acceptor antibody sequence. Thenumber of such substitutions of acceptor for donor amino acids in theCDRs to include reflects a balance of competing considerations. Suchsubstitutions are potentially advantageous in decreasing the number ofmouse amino acids in a humanized antibody and consequently decreasingpotential immunogenicity. However, substitutions can also cause changesof affinity, and significant reductions in affinity are preferablyavoided. Positions for substitution within CDRs and amino acids tosubstitute can also be selected empirically.

The human acceptor antibody sequences can optionally be selected fromamong the many known human antibody sequences to provide a high degreeof sequence identity (e.g., 65%-85% identity) between a human acceptorsequence variable region frameworks and corresponding variable regionframeworks of a donor antibody chain.

Certain amino acids from the human variable region framework residuescan be selected for substitution based on their possible influence onCDR conformation and/or binding to antigen. Investigation of suchpossible influences is by modeling, examination of the characteristicsof the amino acids at particular locations, or empirical observation ofthe effects of substitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid can be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

-   (1) noncovalently binds antigen directly,-   (2) is adjacent to a CDR region,-   (3) otherwise interacts with a CDR region (e.g. is within about 6 Å    of a CDR region), (e.g., identified by modeling the light or heavy    chain on the solved structure of a homologous known immunoglobulin    chain); and-   (4) a residue participating in the VL-VH interface.

Framework residues from classes (1)-(3) as defined by Queen, U.S. Pat.No. 5,530,101 are sometimes alternately referred to as canonical andvernier residues. Framework residues that help define the conformationof a CDR loop are sometimes referred to as canonical residues (Chothiaand Lesk, J. Mol. Biol. 196, 901-917 (1987), Thornton & Martin J. Mol.Biol., 263, 800-815, 1996). Framework residues that supportantigen-binding loop conformations and play a role in fine-tuning thefit of an antibody to antigen are sometimes referred to as vernierresidues (Foote & Winter, 1992, J Mol Bio. 224, 487-499).

Other framework residues that are candidates for substitution areresidues creating a potential glycosylation site. Still other candidatesfor substitution are acceptor human framework amino acids that areunusual for a human immunoglobulin at that position. These amino acidscan be substituted with amino acids from the equivalent position of themouse donor antibody or from the equivalent positions of more typicalhuman immunoglobulins.

The invention provides humanized forms of the mouse 5C1 antibody. Themouse antibody comprises mature heavy and light chain variable regionshaving amino acid sequences comprising SEQ ID NO: 9 and SEQ ID NO: 24,respectively. The invention provides five exemplified humanized matureheavy chain variable regions: H1, SEQ ID NO: 14; H2, SEQ ID NO: 15; H3,SEQ ID NO: 16; H4, SEQ ID NO: 17; and H5, SEQ ID NO: 18. The inventionfurther provides four exemplified humanized mature light chain variableregions: L1, SEQ ID NO: 29; L2, SEQ ID NO: 30; L3, SEQ ID NO: 31; andL4, SEQ ID NO: 32. Antibodies include any permutations of these matureheavy and light chain variable regions are provided, i.e., H1L2, H1L3,H1L4, H2L1, H2L2, H2L3, H2L4, H3L1, H3L2, H3L3, H3L4, H4L1, H4L2, H4L3,H4L4, H5L1, H5L2, H5L3, or H5L4. The H4L3 variant, which includes eightheavy chain backmutations and five light chain backmutations, has anaffinity to α-synuclein (as measured on a Biacore instrument) that iswithin a factor of two of the affinities of the murine and chimeric 5C1antibodies. See Table 3, below. As measured by ELISA, the H4L3 varianthas an affinity for α-synuclein that is substantially the same as achimeric 5C1 antibody (within experimental error) and superior to themurine 5C1 antibody. See FIG. 5. In addition, the H5L3 variant, whichincludes six heavy chain backmutations and five light chainbackmutations, provides an affinity to human α-synuclein (as measured ona Biacore instrument) that is within a factor of four the affinities ofthe murine and chimeric 5C1 antibodies. See Table 3, below. The H3L4variant, which includes nine heavy chain backmutations and two lightchain backmutations, also provides an affinity to human α-synuclein (asmeasured by ELISA) that is substantially the same a chimeric 5C1antibody, within experimental error, and the H3L3 and H3L1 variants,which each include nine heavy chain backmutations and five and six lightchain backmutations, respectively, provide affinities to α-synucleinthat are superior to the murine 5C1 antibody (as measured by ELISA).

The invention provides variants of the H4L3 humanized 5C1 antibody inwhich the humanized mature heavy chain variable region shows at least90%, 95%, 96%, 97%, 98%, or 99% identity to H4 (SEQ ID NO: 17) and thehumanized mature light chain variable region shows at least 90%, 95%,96%, 97%, 98%, or 99% sequence identity to L3 (SEQ ID NO: 31). In somesuch antibodies, at least one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, or all thirteen of the backmutationsin H4L3 are retained. The invention also provides variants of the H5L3humanized 5C1 antibody in which the humanized mature heavy chainvariable region shows at least 90%, 95%, 96%, 97%, 98%, or 99% identityto H5 (SEQ ID NO: 18) and the humanized mature light chain variableregion shows at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identityto L3 (SEQ ID NO: 31). In some such antibodies, at least one, two,three, four, five, six, seven, eight, nine, ten, or all eleven of thebackmutations in H5L3 are retained. The invention also provides variantsof the H3L4 humanized 5C1 antibody in which the humanized mature heavychain variable region shows at least 90%, 95%, 96%, 97%, 98%, or 99%identity to H3 (SEQ ID NO: 16) and the humanized mature light chainvariable region shows at least 90%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to L4 (SEQ ID NO: 32). In some such antibodies, at least one,two, three, four, five, six, seven, eight, nine, ten, or all eleven ofthe backmutations in H3L4 are retained. In some antibodies, at least oneof positions H11, H27, H30, H48, and H73 in the Vh region is occupied byL, Y, T, I, and K, respectively. In some antibodies, positions H11, H27,H30, H48, and H73 in the Vh region are occupied by L, Y, T, I, and K,respectively. In some antibodies, at least one of positions H67, H69,H91, and H94 in the Vh region is occupied by A, L, F, and S,respectively. In some antibodies, positions H67, H69, and H94 in the Vhregion are occupied by A, L, and S, respectively, such as in version H4.In some antibodies, position H94 is occupied by S, such as in versionH5. In some antibodies, positions H67, H69, H91, and H94 in the Vhregion are occupied by A, L, F, and S, respectively, such as in versionH3. In some antibodies, at least one of positions L12 and L14 in the Vkregion is occupied by S. In some antibodies, positions L12 and L14 inthe Vk region are both occupied by S, such as in versions L3 and L4. Insome antibodies, at least one of positions L2, L45, L49, and L87 in theVk region is occupied by V, K, N, and F, respectively. In someantibodies, positions L2, L49, and L87 in the Vk region are occupied byV, N, and F, respectively, such as in version L3. In some antibodies,positions L2, L45, L49, and L87 in the Vk region are occupied by V, K,N, and F, respectively, such as in version L1. The CDR regions of suchhumanized antibodies can be identical or substantially identical to theCDR regions of H4L3 or H5L3, which are the same as those of the mousedonor antibody. The CDR regions can be defined by any conventionaldefinition (e.g., Chothia) but are preferably as defined by Kabat.

One possibility for additional variation in humanized 5C1 variants isadditional backmutations in the variable region frameworks. Many of theframework residues not in contact with the CDRs in the humanized mAb canaccommodate substitutions of amino acids from the correspondingpositions of the donor mouse mAb or other mouse or human antibodies, andeven many potential CDR-contact residues are also amenable tosubstitution or even amino acids within the CDRs may be altered, forexample, with residues found at the corresponding position of the humanacceptor sequence used to supply variable region frameworks. Inaddition, alternate human acceptor sequences can be used, for example,for the heavy and/or light chain. If different acceptor sequences areused, one or more of the backmutations recommended above may not beperformed because the corresponding donor and acceptor residues arealready the same without backmutation. For example, when using a heavychain acceptor sequence in which position H11 is already occupied by L,H48 is already occupied by I, and/or H73 is already occupied by K, thecorresponding backmutation(s) is not necessary. Similarly, when using alight chain acceptor sequence in which position L12 and/or L14 isoccupied by S, the corresponding backmutation(s) is not necessary.

The invention also includes humanized antibodies in which the maturelight and heavy chain variable regions shows at least 90%, 95%, 96%,97%, 98%, or 99% sequence identity to the mature light and heavy chainvariable regions of the humanized 5C1 H1L1, H1L2, H1L3, H1L4, H2L1,H2L2, H2L3, H2L4, H3L1, H3L2, H3L3, H4L1, H4L2, H4L4, H5L1, H5L2, orH5L4. The CDR regions of such humanized antibodies can be identical orsubstantially identical to those of the mouse donor antibody. The CDRregions can be defined by any conventional definition (e.g., Chothia)but are preferably as defined by Kabat.

D. Selection of Constant Region

The heavy and light chain variable regions of chimeric, humanized(including veneered), or human antibodies can be linked to at least aportion of a constant region sufficient to interact with an Fc receptor.The constant region is typically human, but a non-human constant regioncan be selected as needed.

The choice of constant region depends, in part, on whetherantibody-dependent complement and/or cellular mediated cytotoxicity isdesired. For example, human isotopes IgG1 and IgG3 havecomplement-mediated cytotoxicity whereas human isotypes IgG2 and IgG4have poor or no complement-mediated cytotoxicity. A human IgG1 constantregion suitable for inclusion in the antibodies of the invention canhave the sequence of SEQ ID NO: 38. Light chain constant regions can belambda or kappa. A human kappa light chain constant region suitable forinclusion in the antibodies of the invention can have the sequence ofSEQ ID NO: 40. Antibodies can be expressed as tetramers containing twolight and two heavy chains, as separate heavy chains, as separate lightchains, as Fab, Fab′, F(ab′)₂, or Fv fragments, or as single chainantibodies in which heavy and light chain variable regions are linkedthrough a spacer.

Human constant regions show allotypic variation and isoallotypicvariation between different individuals. That is, the constant regionscan differ in different individuals at one or more polymorphicpositions. Isoallotypes differ from allotypes in that sera recognizingan isoallotype binds to a non-polymorphic region of one or more otherisotypes. Reference to a human constant region includes a constantregion with any natural allotype or any permutation of residuesoccupying polymorphic positions in natural allotypes or up to 3, 5 or 10substitutions for reducing or increasing effector function as describedbelow.

One or several amino acids at the amino or carboxy terminus of the lightand/or heavy chain, such as the C-terminal lysine of the heavy chain,may be missing or derivatized in a proportion or all of the molecules.Substitutions can be made in the constant regions to reduce or increaseeffector function such as complement-mediated cytotoxicity or ADCC (see,e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No.5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006),or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol.Chem. 279:6213, 2004). Exemplary substitutions include a Gln at position250 and/or a Leu at position 428 (EU numbering is used in this paragraphfor the constant region) for increasing the half-life of an antibody.Substitution at any or all of positions 234, 235, 236 and/or 237 reducesaffinity for Fcy receptors, particularly FcyRI receptor (see, e.g., U.S.Pat. No. 6,624,821). An alanine substitution at positions 234, 235 and237 of human IgG1 can be used for reducing effector functions.Optionally, positions 234, 236 and/or 237 in human IgG2 are substitutedwith alanine and position 235 with glutamine. (See, e.g., U.S. Pat. No.5,624,821). In some aspects, a mutation at one or more of positions 241,264, 265, 270, 296, 297, 322, 329, and 331 by EU numbering of human IgG1is used. In some aspects, a mutation at one or more of 318, 320, and 322by EU numbering of human IgG1 is used. In some aspects, the isotype ishuman IgG2 or IgG4.

E. Human Antibodies

Human antibodies against α-synuclein are provided by a variety oftechniques described below. Some human antibodies are selected bycompetitive binding experiments, or otherwise, to have the same oroverlapping epitope specificity as 5C1. Human antibodies can also bescreened for a particular epitope specificity by using only a fragmentof α-synuclein (e.g., amino acid residues 118-126) as the immunogen,and/or by screening antibodies against a collection of deletion mutantsof α-synuclein. One technique for producing human antibodies is triomamethodology (Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S.Pat. No. 4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666).Another technique involves immunizing transgenic mice expressing humanimmunoglobulin genes, such as the XenoMouse®, AlivaMab Mouse orVeloceimmune mouse (see, e.g., Lonberg et al., W093/1222, U.S. Pat. No.5,877,397, U.S. Pat. No. 5,874,299, U.S. Pat. No. 5,814,318, U.S. Pat.No. 5,789,650, U.S. Pat. No. 5,770,429, U.S. Pat. No. 5,661,016, U.S.Pat. No. 5,633,425, U.S. Pat. No. 5,625,126, U.S. Pat. No. 5,569,825,U.S. Pat. No. 5,545,806, Nature 148, 1547-1553 (1994), NatureBiotechnology 14, 826 (1996), Kucherlapati, and WO 91/10741). Anothertechnique is phage display (see, e.g., Dower et al., WO 91/17271 andMcCafferty et al., WO 92/01047, U.S. Pat. No. 5,877,218, U.S. Pat. No.5,871,907, U.S. Pat. No. 5,858,657, U.S. Pat. No. 5,837,242, U.S. Pat.No. 5,733,743 and U.S. Pat. No. 5,565,332). In these methods, librariesof phage are produced in which members display different antibodies ontheir outer surfaces. Antibodies are usually displayed as Fv or Fabfragments. Phage displaying antibodies with a desired specificity areselected by affinity enrichment to an α-synuclein peptide or fragmentthereof. Another technique is to sequence DNA from human B cellsaccording to the general protocols outlined in Reddy et al., NatBiotechnol. 2010 Sep. 28 (9):965-9 (Epub 2010 Aug. 29), and US20110053803, 20100099103, 20100291066, 20100035763, and 20100151471.Briefly, B cells can be obtained from a human suspected of havinganti-α-synuclein antibodies, e.g., a human immunized with α-synuclein,fragments thereof, longer polypeptides containing α-synuclein orfragments thereof, or anti-idiotypic antibodies. The mRNA of theantibodies from B cells is then reverse transcribed into cDNA andsequenced using, e.g., 454 sequencing technology. After obtaining thesequences of the chains from each antibody, the chains can be pairedtogether (e.g., using bioinformatics), cloned, expressed, and screenedfor desired properties.

F. Expression of Recombinant Antibodies

A number of methods are known for producing chimeric and humanizedantibodies using an antibody-expressing cell line (e.g., hybridoma). Forexample, the immunoglobulin variable regions of antibodies can be clonedand sequenced using well known methods. In one method, the heavy chainvariable VH region is cloned by RT-PCR using mRNA prepared fromhybridoma cells. Consensus primers are employed to VH region leaderpeptide encompassing the translation initiation codon as the 5′ primerand a g2b constant regions specific 3′ primer. Exemplary primers aredescribed in U.S. patent publication US 2005/0009150 by Schenk et al.(hereinafter, “Schenk”). The sequences from multiple,independently-derived clones, can be compared to ensure no changes areintroduced during amplification. The sequence of the VH region can alsobe determined or confirmed by sequencing a VH fragment obtained by 5′RACE RT-PCR methodology and the 3′ g2b specific primer.

The light chain variable VL region can be cloned in an analogous manneras the VH region. In one approach, a consensus primer set designed foramplification of VL regions is designed to hybridize to the VL regionencompassing the translation initiation codon, and a 3′ primer specificfor the Ck region downstream of the V-J joining region. In a secondapproach, 5′RACE RT-PCR methodology is employed to clone a VL encodingcDNA. Exemplary primers are described in Schenk, supra. The clonedsequences are then combined with sequences encoding human (or othernon-human species) constant regions. Exemplary sequences encoding humanconstant regions include SEQ ID NO: 37, which encodes a human IgG1constant region, and SEQ ID NO: 39, which encodes a human kappa lightchain constant region.

In one approach, the heavy and light chain variable regions arere-engineered to encode splice donor sequences downstream of therespective VDJ or VJ junctions, and cloned into the mammalian expressionvector, such as pCMV-hγ1 for the heavy chain, and pCMV-Mc1 for the lightchain. These vectors encode human γ1 and Ck constant regions as exonicfragments downstream of the inserted variable region cassette. Followingsequence verification, the heavy chain and light chain expressionvectors can be co-transfected into CHO cells to produce chimericantibodies. Conditioned media is collected 48 hrs. post-transfection andassayed by western blot analysis for antibody production or ELISA forantigen binding. The chimeric antibodies are humanized as describedabove.

Chimeric, veneered, humanized, and human antibodies are typicallyproduced by recombinant expression. Recombinant nucleic acid constructstypically include an expression control sequence operably linked to thecoding sequences of antibody chains, including naturally associated orheterologous expression control element(s), such as a promoter. Once thevector has been incorporated into the appropriate host, the host ismaintained under conditions suitable for high level expression of thenucleotide sequences, and the collection and purification of the crossreacting antibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers, e.g.,ampicillin-resistance or hygromycin-resistance, to permit detection ofthose cells transformed with the desired DNA sequences.

E. coli is one prokaryotic host useful for cloning the DNA sequencesencoding the polypeptides disclosed herein. Microbes, such as yeast arealso useful for expression. Saccharomyces is a yeast host, with suitablevectors having expression control sequences, an origin of replication,termination sequences and the like as desired. Typical promoters include3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeastpromoters include, among others, promoters from alcohol dehydrogenase,isocytochrome C, and enzymes responsible for maltose and galactoseutilization.

Mammalian cells are a host cell for expressing nucleotide segmentsencoding immunoglobulins or fragments thereof. See Winnacker, From Genesto Clones, (VCH Publishers, NY, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped, and include CHO cell lines, various COS cell lines, HeLacells, L cells, human embryonic kidney cell, and myeloma cell lines. Thecells can be nonhuman. Expression vectors for these cells can includeexpression control sequences, such as an origin of replication, apromoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), andnecessary processing information sites, such as ribosome binding sites,RNA splice sites, polyadenylation sites, and transcriptional terminatorsequences. Expression control sequences can include promoters derivedfrom endogenous genes, cytomegalovirus, SV40, adenovirus, bovinepapillomavirus, and the like. See Co et al., J. Immunol. 148:1149(1992).

Alternatively, antibody coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,U.S. Pat. No. 5,741,957, U.S. Pat. No. 5,304,489, U.S. Pat. No.5,849,992). Suitable transgenes include coding sequences for lightand/or heavy chains in operable linkage with a promoter and enhancerfrom a mammary gland specific gene, such as casein or betalactoglobulin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by methods depending on the type of cellular host.For example, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment, electroporation,lipofection, biolistics or viral-based transfection can be used forother cellular hosts. Other methods used to transform mammalian cellsinclude the use of polybrene, protoplast fusion, liposomes,electroporation, and microinjection. For production of transgenicanimals, transgenes can be microinjected into fertilized oocytes, or canbe incorporated into the genome of embryonic stem cells, and the nucleiof such cells transferred into enucleated oocytes.

Having introduced vector(s) encoding antibody heavy and light chainsinto cell culture, cell pools can be screened for growth productivityand product quality in serum-free media. Top-producing cell pools canthen be subjected of FACS-based single-cell cloning to generatemonoclonal lines. Specific productivities above 50 pg or 100 pg per cellper day, which correspond to product titers of greater than 7.5 g/Lculture, can be used. Antibodies produced by single cell clones can alsobe tested for turbidity, filtration properties, PAGE, IEF, UV scan,HP-SEC, carboydrate-oligosaccharide mapping, mass spectrometery, andbinding assay, such as ELISA or Biacore. A selected clone can then bebanked in multiple vials and stored frozen for subsequent use.

Once expressed, antibodies can be purified according to standardprocedures of the art, including protein A capture, HPLC purification,column chromatography, gel electrophoresis and the like (see generally,Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

Methodology for commercial production of antibodies can be employed,including codon optimization, selection of promoters, transcriptionelements, and terminators, serum-free single cell cloning, cell banking,use of selection markers for amplification of copy number, CHOterminator, serum free single cell cloning, improvement of proteintiters (see, e.g., U.S. Pat. No. 5,786,464, U.S. Pat. No. 6,114,148,U.S. Pat. No. 6,063,598, U.S. Pat. No. 7,569,339, W02004/050884,W02008/012142, W02008/012142, W02005/019442, W02008/107388, andW02009/027471, and U.S. Pat. No. 5,888,809).

G. Antibody Screening Assays

Antibodies can be subject to several screens including binding assays,functional screens, screens in animal models of diseases associated withα-synuclein deposits, and clinical trials. Binding assays test forspecific binding and, optionally, affinity and epitope specificity toα-synuclein (or a fragment thereof, such as amino acid residues118-126). Such screens are sometimes performed in competition with anexemplary antibody such as 5C1. Optionally, either the antibody orα-synuclein target is immobilized in such assay. Functional assays canbe performed in cellular models including cells naturally expressingα-synuclein or transfected with DNA encoding α-synuclein or a fragmentthereof. Suitable cells include neuronal cells. Cells can be screenedfor reduced levels of α-synuclein (e.g., by Western blotting orimmunoprecipitation of cell extracts or supernatants), reduced levels ofaggregated α-synuclein (e.g., by immunohistochemical and/or confocalmethods), and/or reduced toxicity attributable to α-synuclein.

Animal model screens test the ability of the antibody to therapeuticallyor prophylactically treat signs or symptoms in an animal modelsimulating a human disease associated with α-synuclein deposits, such asa Lewy Body disease. Suitable signs or symptoms that can be monitoredinclude motor balance, coordination, and cognitive deficits. The extentof impairment can be determined by comparison with an appropriatecontrol, such as motor balance, coordination, or cognitive deficiency incontrol animals that have received a control antibody (e.g., an isotypematched control antibody), a placebo, or no treatment at all. Transgenicor other animal models of Lewy Body diseases can express a humanα-synuclein transgene. To facilitate testing in animal models,antibodies having a constant region appropriate for the animal model canbe used. It can be concluded that a humanized version of an antibodywill be effective if the corresponding mouse antibody or chimericantibody is effective in an appropriate animal model and the humanizedantibody has similar binding affinity (e.g., by a factor of 1.5, 2, or3, within experimental error).

Clinical trials test for safety and efficacy in a human having a diseaseassociated with α-synuclein deposits.

H. Nucleic Acids

The invention further provides nucleic acids encoding any of the heavyand light chains described above. Typically, the nucleic acids alsoencode a signal peptide fused to the mature heavy and light chains.Suitable example of signal peptides include amino acid residues 1-19 ofSEQ ID NO: 6 (encoded by nucleotides 1-57 of SEQ ID NO: 5) and aminoacid residues 1-19 of SEQ ID NO: 8 (encoded by nucleotides 1-57 of SEQID NO: 7). Coding sequences on nucleic acids can be in operable linkagewith regulatory sequences to ensure expression of the coding sequences,such as a promoter, enhancer, ribosome binding site, transcriptiontermination signal and the like. The nucleic acids encoding heavy andlight chains can occur in isolated form or can be cloned into one ormore vectors. The nucleic acids can be synthesized by for example, solidstate synthesis or PCR of overlapping oligonucleotides. Nucleic acidsencoding heavy and light chains can be joined as one contiguous nucleicacid, e.g., within an expression vector, or can be separate, e.g., eachcloned into its own expression vector.

V. Therapeutic Applications

The invention provides several methods of treating or effectingprophylaxis of Lewy Body diseases in patients suffering from or at riskof such a disease. Patients amenable to treatment include individuals atrisk of disease of a LBD but not showing symptoms, as well as patientspresently showing symptoms or the early warning signs ofsynucleinopathies, for example, EEG slowing, neuropsychiatricmanifestations (depression, dementia, hallucinations, anxiety, apathy,anhedonia), autonomic changes (orthostatic hypotension, bladderdisturbances, constipation, fecal incontinence, sialorrhea, dysphagia,sexual dysfunction, changes in cerebral blood flow), sensory changes(olfactory, pain, color discrimination abnormal sensations), sleepdisorders (REM sleep behavior disorder (RBD), restless legssyndrome/periodic extremity movements, hypersomnia, insomnia) andmiscellaneous other signs and symptoms (fatigue, diplopia, blurredvision, seborrhea, weight loss/gain). Therefore, the present methods canbe administered prophylactically to individuals who have a known geneticrisk of a LBD. Such individuals include those having relatives who haveexperienced this disease, and those whose risk is determined by analysisof genetic or biochemical markers. Genetic markers of risk toward PDinclude mutations in the α-synuclein or Parkin, UCHLI, and CYP2D6 genes;particularly mutations at positions 30 and 53 of the α-synuclein gene.Individuals presently suffering from Parkinson's disease can berecognized from its clinical manifestations including resting tremor,muscular rigidity, bradykinesia and postural instability.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by assayingantibody, or activated T-cell or B-cell responses to a therapeutic agent(e.g., a truncated form of α-synuclein peptide) over time. If theresponse falls, a booster dosage is indicated.

The invention provides methods of treating or effecting prophylaxis ofLewy Body disease in a patient by administration of antibodycompositions under conditions that generate a beneficial therapeuticresponse in the patient (e.g., reduction of neuritic and/or axonal alphasynuclein aggregates, reduction of neuritic dystrophy, improvingcognitive function, and/or reversing, treating or inhibiting cognitivedecline) in the patient. In some methods, the areas of neuriticdystrophy in the neuropil of neocortex and/or basal ganglia can bereduced by 10%, 20%, 30%, 40% or more as compared to a control.

Cognitive impairment, progressive decline in cognitive function, changesin brain morphology, and changes in cerebrovascular function arecommonly observed in patients suffering from or at risk of Lewy Bodydisease. The invention provides methods of inhibiting decline ofcognitive function in such patients.

The invention also provides methods of preserving or increasing synapticdensity and/or dentritic density. An index of changes in synaptic ordentritic density can be measured by markers of synapse formation(synaptophysin) and/or dendrites (MAP2). In some methods, the synapticor dentritic density can be restored to the level of synaptic ordentritic density in a healthy subject. In some methods, the level ofsynaptic or dentritic density in a patient can be elevated by 5%, 10%,15%, 20%, 25%, 30% or more as compared to a control.

VI. Pharmaceutical Compositions and Methods Of Treatment

In prophylactic applications, an antibody or a pharmaceuticalcomposition the same is administered to a patient susceptible to, orotherwise at risk of a disease in a regime (dose, frequency and route ofadministration) effective to reduce the risk, lessen the severity, ordelay the onset of at least one sign or symptom of the disease. In someprophylactic applications, the regime is effective to inhibit or delayaccumulation of alpha synuclein and truncated fragments in the brain,and/or inhibit or delay its toxic effects and/or inhibit/or delaydevelopment of behavioral deficits. In therapeutic applications, anantibody is administered to a patient suspected of, or already sufferingfrom a Lewy body disease in a regime (dose, frequency and route ofadministration) effective to ameliorate or at least inhibit furtherdeterioration of at least one sign or symptom of the disease. In sometherapeutic applications, the regime is effective to reduce or at leastinhibit further increase of levels of alpha synuclein and truncatedfragments, associated toxicities and/or behavioral deficits.

A regime is considered therapeutically or prophylactically effective ifan individual treated patient achieves an outcome more favorable thanthe mean outcome in a control population of comparable patients nottreated by methods of the invention, or if a more favorable outcome isdemonstrated in treated patients versus control patients in a controlledclinical trial (e.g., a phase II, phase II/III or phase III trial) atthe p<0.05 or 0.01 or even 0.001 level.

Effective doses vary depending on many different factors, includingmeans of administration, target site, physiological state of the patientincluding type of Lewy body disease, whether the patient is an ApoEcarrier, whether the patient is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic.

An exemplary dosage range for antibodies is from about 0.01 to 5 mg/kg,and more usually 0.1 to 3 mg/kg or 0.15-2 mg/kg or 0.15-1.5 mg/kg, ofpatient body weight. Antibody can be administered such doses daily, onalternative days, weekly, fortnightly, monthly, quarterly, or accordingto any other schedule determined by empirical analysis. An exemplarytreatment entails administration in multiple dosages over a prolongedperiod, for example, of at least six months. Additional exemplarytreatment regimes entail administration once per every two weeks or oncea month or once every 3 to 6 months.

Antibodies can be administered via a peripheral route (i.e., one inwhich an administered or induced antibody crosses the blood brainbarrier to reach an intended site in the brain. Routes of administrationinclude topical, intravenous, oral, subcutaneous, intraarterial,intracranial, intrathecal, intraperitoneal, intranasal or intramuscular.Some routes for administration of antibodies are intravenous andsubcutaneous. This type of injection is most typically performed in thearm or leg muscles. In some methods, antibodies are injected directlyinto a particular tissue where deposits have accumulated, for exampleintracranial injection.

Pharmaceutical compositions for parenteral administration are can besterile and substantially isotonic and manufactured under GMPconditions. Pharmaceutical compositions can be provided in unit dosageform (i.e., the dosage for a single administration). Pharmaceuticalcompositions can be formulated using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries. Theformulation depends on the route of administration chosen. Forinjection, antibodies can be formulated in aqueous solutions, preferablyin physiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline or acetate buffer (to reducediscomfort at the site of injection). The solution can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively antibodies can be in lyophilized form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The present regimes can be administered in combination with anotheragent effective in treatment or prophylaxis of the disease beingtreated. For example, in the case of Parkinson's disease, immunotherapyagainst alpha synuclein WO/2008/103472, Levodopa, dopamine agonists,COMT inhibitors, MAO-B inhibitors, Amantadine, or anticholinergic agentscan be used in combination with the present regimes.

VII. Other Applications

The antibodies described above can be used for detecting α-synuclein inthe context of clinical diagnosis or treatment or in research. Theantibodies can also be sold as research reagents for laboratory researchin detecting cells bearing α-synuclein and their response to variousstimuli. In such uses, monoclonal antibodies can be labeled withfluorescent molecules, spin-labeled molecules, enzymes or radioisotypes,and can be provided in the form of kit with all the necessary reagentsto perform the assay for α-synuclein. The antibodies can also be used topurify α-synuclein, e.g., by affinity chromatography.

The antibodies can be used for detecting LBs in a patient. Such methodsare useful to diagnose or confirm diagnosis of PD, or other diseaseassociated with the presence of LBs in the brain, or susceptibilitythereto. For example, the methods can be used on a patient presentingwith symptoms of dementia. If the patient has LBs, then the patient islikely suffering from a Lewy body disease, such as Parkinson's disease.The methods can also be used on asymptomatic patients. Presence of Lewybodies or other abnormal deposits of α-synuclein indicatessusceptibility to future symptomatic disease. The methods are alsouseful for monitoring disease progression and/or response to treatmentin patients who have been previously diagnosed with a Lewy body disease.

The methods can be performed by administering an antibody and thendetecting the antibody after it has bound. If desired, the clearingresponse can be avoided by using an antibody fragment lacking afull-length constant region, such as a Fab. In some methods, the sameantibody can serve as both a treatment and diagnostic reagent.

For diagnosis (e.g., in vivo imaging), the antibodies can beadministered by intravenous injection into the body of the patient, ordirectly into the brain by intracranial injection or by drilling a holethrough the skull. The dosage of reagent should be within the sameranges as for treatment methods. Typically, the antibody is labeled,although in some methods, the antibody is unlabeled and a secondarylabeling agent is used to bind to the antibody. The choice of labeldepends on the means of detection. For example, a fluorescent label issuitable for optical detection. Use of paramagnetic labels is suitablefor tomographic detection without surgical intervention. Radioactivelabels can also be detected using PET or SPECT.

Diagnosis is performed by comparing the number, size and/or intensity oflabeled loci to corresponding base line values. The base line values canrepresent the mean levels in a population of undiseased individuals.Base line values can also represent previous levels determined in thesame patient. For example, base line values can be determined in apatient before beginning treatment, and measured values thereaftercompared with the base line values. A decrease in values relative tobase line signals a positive response to treatment.

The antibodies can be used to generate anti-idiotype antibodies. (see,e.g., Greenspan & Bona, FASEB J. 7 (5):437-444, 1989; and Nissinoff, J.Immunol. 147:2429-2438, 1991). Such anti-idiotype antibodies can beutilized in pharmacokinetics, pharmacodynamics, biodistribution studiesas well as in studies of clinical human-anti-human antibody (HAHA)responses in individuals treated with the antibodies. For example,anti-idiotypic antibodies bind specifically the variable region ofhumanized 5C1 antibodies and therefore can be used to detect humanized5C1 antibodies in pharmacokinetic studies and help to quantifyhuman-anti-human antibody (HAHA) responses in treated individuals.

VIII. Kits

Also provided are kits including an α-synuclein-specific antibody andinstructions for use. Such kits can be used for, e.g., performing thediagnostic methods described above. A kit can also include a label. Kitsalso typically contain labeling providing directions for use of the kit.The labeling may also include a chart or other correspondence regimecorrelating levels of measured label with levels of antibodies toα-synuclein. The term labeling generally refers to any written orrecorded material that is attached to, or otherwise accompanies a kit atany time during its manufacture, transport, sale or use. For example,the term labeling encompasses advertising leaflets and brochures,packaging materials, instructions, audio or video cassettes, computerdiscs, as well as writing imprinted directly on kits.

Also provided are diagnostic kits for performing in vivo imaging. Suchkits typically contain an antibody binding to an epitope of α-synucleinas described herein. The antibody can be labeled or a secondary labelingreagent is included in the kit. The kit can include instructions forperforming an in vivo imaging assay.

All patent filings, website, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

EXAMPLES Example 1 Isolation of Murine 5C1

The murine 5C1 antibody was generated in a mouse injected with a peptideconjugate containing the peptide immunogen VDPDNEAYEGGC (SEQ ID NO: 14)coupled to a sheep anti- mouse antibody. The peptide, which includesresidues 118-126 of α-synuclein fused to a C-terminal GGC peptide, wascoupled to the sheep anti-mouse antibody via a maleimide linker bound tothe C-terminal cysteine residue.

Example 2 Passive Immunization with α-Synuclein Antibodies

To test the effect of α-synuclein antibodies on an animal model for LewyBody disease, various α-synuclein antibodies were used to passivelyimmunize mice. 3-4 month old wildtype, α-synuclein knockout, andα-synuclein transgenic (line 61) female mice were used (n=14/group).Antibodies that were tested included:

-   9E4 (IgG1, epitope: amino acids 118-126 of α-synuclein);-   5C1 (IgG1, immunogen: amino acids 118-126 of α-synuclein,    cys-linker);-   5D12(IgG2, immunogen: amino acids 118-126 of α-synuclein, n-linker);-   1H7 (IgG1, epitope: amino acids 91-99 of α-synuclein); and-   27-1 (IgG1 control antibody).

Mice received an antibody dosage of 10 mg/kg over a 5 month period, fora total of 21 injections. In addition, the animals were injected withlentivirus (LV) expressing human α-synuclein (wt) by unilateralintroduction of human α-synuclein (wt) into the hippocampus.

Readout antibodies included α-synuclein antibodies from Chemicon(epitope: full-length α-synuclein), Millipore (epitope: full-lengthα-synuclein), and ELADW 105 (epitope: amino acids 121-124 ofα-synuclein, preferably with α-synuclein truncated at residue 122-124).

Endpoints: Antibody titers were monitored prior to termination of theexperiment. Behavior was assessed using the Morris Water Maze (MWM) andhorizontal round beam tests. The round beam test assesses motor balance,coordination, and gait using two beams of varying diameter. Beam A (thetraining beam) is larger in diameter, and therefore easier to traverse.Beam D (the testing beam) is smaller in diameter, and therefore moredifficult to traverse. Water maze performance was carried out at weeks10 and just prior to termination. On termination of the experiment, micewere sacrificed and neuropathology measurements were obtained forα-synuclein aggregation, synaptophysin, and MAP2. In addition,biochemistry measurements were obtained for α-synuclein, PSD95, andsynaptophysin. Selected multilabeling and confocal labeling were carriedout using synaptic, neuronal and glial markers.

Results: The results showed that all antibodies, except 5D12, producedsignificant reduction in α-syn accumulation and preservation of synapticand dendritic densities, as well as positive outcomes in MWMperformance. The 9E4 antibody was effective in in vitro and in vivostudies as well as behavioral assays. In particular, the resultsindicate that α-synuclein antibodies may reduce neuritic/axonalα-synuclein aggregates.

Behavioral Results: The 5C1 and 9E4 antibodies improved water mazeperformance in α-synuclein transgenic mice, as did 1H7, albeit to alesser extent. See FIG. 3. In contrast, the 5D12 antibody did notimprove water maze performance in α-synuclein transgenic mice. Withregard to the horizontal round beam test, the 9E4 and 1H7 antibodiesimproved performance as measured both by speed and number of errors,whereas the 5D12 and 5C1 antibodies did not. See FIG. 4. The data inFIG. 4 is presented as the number of slips/10 cm (i.e., “errors”) andthe ratio of distance traveled divided by time taken to travel thedistance (i.e., “speed,” measured in units of 10 cm/sec).

Neuropathology Results: The 5C1, 9E4, and 1H7 antibodies reducedELADW-105 positive neuritic dystrophy, whereas the 5D12 antibody didnot. In α-synuclein transgenic mice, the 9E4 antibody reduced the areaof neuropil by 43% in the neocortex and by 40% in the basal ganglia, ascompared to control mice (i.e., mice receiving the 27-1 IgG1 controlantibody). The 9E4 antibody also preserved staining for synaptophysinand MAP2 in the neocortex and basal ganglia.

Example 3 Sequencing of Variable Domains of 5C1

mRNA was extracted and purified from a 5C1 hybridoma cell pellet usingQIAGEN® OLIGOTEX® mRNA kit. Purified mRNA was next transcribed into cDNAusing an oligo dT anti-sense primer and the INVITROGEN® SUPERSCRIPT® IIkit. Nucleic acid sequences coding for the 5C1 heavy chain and lightchain variable regions were amplified from the cDNA by PCR, usingdegenerate VH and VL sense primers and a gene-specific (CH/CL)anti-sense primer. The PCR products, which were designed to include thesequence of the signal peptide, variable domain, and constant domain (upto the anti-sense primer), were gel-purified, cloned into a blunt vectoror TA vector, and then sequenced. Sequences were deduced from analysisof at least 3 independent clones having an open reading frame startingwith a methionine and extending through the variable region into theconstant region.

Nucleic acid encoding the 5C1 heavy chain variable region has thesequence of SEQ ID NO: 5. The corresponding protein sequence (FIG. 1),which includes a signal peptide at positions 1-19 (underlined) is asfollows:

(SEQ ID NO: 6) MERHWIFLFLLSVTGGVHSQVQLQQSGAELAKPGTSVQMSCKASGYTFTNYWMNWIKARPGQGLEWIGATNPNNGYTDYNQRFKDKAILTADKSSNTAYMHLSSLTSEDSAVYFCASGGHLAYWGQGTVVTVSA

Nucleic acid encoding the 5C1 light chain variable region has thesequence of SEQ ID NO: 7. The corresponding protein sequence (FIG. 2),which includes a signal peptide at positions 1-19 (underlined) is asfollows:

(SEQ ID NO: 8) MKLPVRLLVLMFWIPASSSDVVMTQIPLYLSVSPGDQASISCRSSQSLFHSKGNTYLHWYLQKPGQSPKLLINRVSNRFSGVPDRFSGSGSGTDFTLKISGVEAEDLGVYFCSQSAHVPWTFGGGTKLEIR

The amino acid sequence for the mature 5C1 heavy chain variable region(SEQ ID NO: 9) is shown in Table 1 (below), and the corresponding aminoacid sequence for the mature 5C1 light chain variable region (SEQ ID NO:24) is shown in Table 2 (below). Kabat numbering is used throughout.

Example 4 Humanization of Murine 5C1

Analysis of the CDRs of the 3H6 Vh region reveals a 5 residue CDR-H1(SEQ ID NO: 10), a 17 residue CDR-H2 (SEQ ID NO: 11), and a 6 residueCDR-H3 (SEQ ID NO: 12). Similar analysis of the CDRs of the 3H6 Vkregion reveals a 16 residue CDR-L1 (SEQ ID NO: 25), a 7 residue CDR-L2(SEQ ID NO: 26), and a 9 residue CDR-L3 (SEQ ID NO: 27).

Analysis of the residues at the interface between the 5C1 Vk and Vhregions reveals that most of the residues are the ones commonly found.

A search of the non-redundant protein sequence database from NCBIallowed selection of suitable human frameworks into which to graft the5C1 murine CDRs. For Vk, a human kappa light chain with NCBI accessioncode CAB51293.1 (GI:5578786; SEQ ID NO: 28) was chosen. For Vh, human Igheavy chain AAY42876.1 (GI:66096557; SEQ ID NO: 13) was chosen.

Exemplary humanized Vh and Vk designs, with backmutations based on theselected human frameworks, are shown in Table 1 and Table 2,respectively.

Exemplary Humanized Vh Designs

Five different humanized versions of the 5C1 Vh region were designed,H1, H2, H3, H4, and H5. In selecting backmutations, residues H11, H27,H30, H48, H67, H69, H73, H91, and H94 were ultimately focused on. Ineach of the humanized Vh region designs, residues H11, H27, H30, H48,and H73 were backmutated to L, Y, T, I, and K, respectively, because theresidues formed part of CDR-H1 according to the Chothia definition (H27and H30) or the corresponding residues in the human framework sequenceare low frequency residues (V at position H11, M at position H48, and Eat position H73). For version H1 (SEQ ID NO: 14), additional residuesH67 and H69 were backmutated (to A and L, respectively) to preserve CDRpacking. In version H2 (SEQ ID NO: 15), no further backmutations wereintroduced (i.e., the backmutations at positions H67 and H69 in versionH1 were eliminated). In version H3 (SEQ ID NO: 16), additional residuesH67, H69, H91, and H94 were backmutated (to A, L, F, and S,respectively). The H67, H69, and H94 backmutations were to preserve CDRpacking, while H91, a Vh/Vk interface residue, was backmutated to testits impact on the interface. In version H4 (SEQ ID NO: 17), additionalresidues H67, H69, and H94 were backmutated (to A, L, and S,respectively). Thus, version H4 differs from H3 in that the backmutationat H91 is eliminated. In version H5 (SEQ ID NO: 18), additional residueH94 was also backmutated (to S), to preserve CDR packing.

Exemplary nucleic acid sequences encoding humanized 5C1 H1, H2, H3, H4,and H5 are provided in SEQ ID NOs: 19, 20, 21, 22, and 23, respectively.

Exemplary Humanized Vk Designs

Four different humanized versions of the 5C1 Vk region were designed,L1, L2, L3, and L4. In selecting backmutations, residues L2, L12, L14,L45, L49, and L87 were ultimately focused on. In each of the humanizedVk region designs, residues L12 and L14 were backmutated to S becausethe corresponding residues in the human framework sequence (P and T,respectively) are low frequency residues. For version L1 (SEQ ID NO:29), additional residues L2, L45, L49, and L87 were backmutated (to V,K, N, and F, respectively). L2 is a canonical/CDR interacting residue;L45 undergoes a polarity/charge switch from the murine to humanframework sequences (K to Q), and thus could impact folding; L49 is aVernier residue; and L87 is a Vh/Vk interface residue. In version L2(SEQ ID NO: 30), additional residue L45 was backmutated to K. Thus,relative to L1, the backmutations at residues L2, L49, and L87 wereeliminated. In version L3 (SEQ ID NO: 31), additional residues L2, L49,and L87 were backmutated (to V, N, and F, respectively). Thus, relativeto L1, the backmutation at residue L45 was eliminated. In version L4(SEQ ID NO: 32), no additional residues were backmutated (i.e., onlyresidues L12 and L14 were backmutated).

Exemplary nucleic acid sequences encoding humanized 5C1 L1, L2, L3, andL4 are provided in SEQ ID NOs: 33, 34, 35, and 36, respectively.

Example 5 Affinity of Humanized 5C1 Antibodies for Alpha-Synuclein

The affinity of various combinations of 5C1 humanized heavy chains andhumanized light chain proteins for α-synuclein was analyzed by ELISA. Asshown in FIG. 5, the H1L1 version of humanized 5C1 antibody displayed noaffinity for α-synuclein under the assay conditions. In contrast, thechimeric 5C1 antibody had a higher affinity for α-synuclein than themurine 5C1 antibody. Humanized versions H3L4, H4L3, and chimeric H+L3performed comparably, and almost as well as the chimeric 5C1 antibody.In addition, humanized versions H3L3 and H3L1 performed comparably,though with slightly lower affinity than H3L4, H4L3, and chimeric H+L3.

Various humanized 5C1 antibody versions were also analyzed by Biacore,to more precisely determine binding affinities. An anti-human IgG CMSBiacore chip was prepared following the protocol supplied by GEHealthcare. Each humanized 5C1 antibody version was independentlycaptured to a level were R_(max) would not exceed 50, using theequation:

R _(max)=(RU of captured antibody)*(MW of Synuclein)/(MW of capturedantibody)*2

The factor of 2 in the denominator is for the number of binding sites onthe antibody. Alpha synuclein was flowed over the chop at aconcentration varied from ˜10× above the expected KD to ˜10× below theexpected KD. Data was collected and double reference subtracted toaccount for drift and a small amount of nonspecific binding. The datawas analyzed using BIAcore evaluation software using a 1:1 model and aglobal fit.

The results of the Biacore analysis are summarized in Table 3 (below).The data indicates that most of the loss in affinity for alpha synucleinis due to an increased off rate in some of the antibody versions. Basedon the affinity data, H4L3 was identified as a preferred antibody.

TABLE 3 Biacore-Determined Affinities of 5C1 Variant Antibodies 5C1 #Framework Mouse AAs Variant HC LC K_(D) K_(on) K_(off) m5C1 82 80  68.7nM 7.5 × 10⁴/s  5.1 × 10³/s Ch5C1 82 80  86.0 nM 6.1 × 10⁴/s  5.3 ×10³/s h5C1_H3L4 65, incl. 9 69, incl. 2 1237.0 nM  4.4 × 10⁴/s 54.5 ×10³/s backmutations backmutations (V11L, G27Y, (P12S, T14S) N30T, M48I,V67A, I69L, E73K, Y91F, R94S) h5C1_H4L3 64, incl. 8 72, incl. 5 119.8 nM4.4 × 10⁴/s  5.1 × 10³/s backmutations backmutations (V11L, G27Y, (I2V,P12S, T14S, N30T, M48I, Y49N, Y87F) h5C1_H4L4 V67A, I69L, 69, incl. 2600.9 nM 5.3 × 10⁴/s 32.4 × 10³/s E73K, R94S) backmutations (P12S, T14S)h5C1_H5L3 62, incl. 6 72, incl. 5 283.1 nM 3.9 × 10⁴/s 11.1 × 10³/sbackmutations backmutations (V11L, G27Y, (I2V, P12S, T14S, N30T, M48I,Y49N, Y87F) h5C1_H5L4 E73K, R94S) 69, incl. 2 1062.0 nM  3.7 × 10⁴/s40.3 × 10³/s backmutations (P12S, T14S)

Example 6 Alanine Scanning Mutagenesis

The epitopes bound by antibodies 5C1, 9E4 and 5D12 have beenapproximately mapped to being within residues 118-126 of alpha synucleindue to the antibodies binding to overlapping peptides. This exampledescribes a more precise mapping, by alanine scanning mutagenesis, ofeach residue between positions 118 and 126 of alpha synuclein. Alanineis used because of its non-bulky, chemically inert, methyl functionalgroup that nevertheless mimics the secondary structure preferences thatmany of the other amino acids possess. The upper portions of FIGS. 6, 7and 8 show the results of Western blots stained with antibodies 9E4, 5C1and 5D12, respectively. The blots include full-length alpha synucleinand point mutants of alpha synuclein produced by alanine scanningmutagenesis of residues 118-126 and were stained with 0.5 μg/ml ofantibody. Mutations at positions 122 and 125 essentially abolish bindingof 9E4, whereas mutations at other positions have little if any effect.Thus, 9E4 predominantly contacts residues 122 and 125. Mutations atpositions 120-122 essentially abolish binding of 5C1, and mutations atpositions 123 and 124 substantially reduce but do not abolish binding.Thus, 5C1 predominantly contacts residues 120-122 and, to a lesserextent, residues 123-124. Mutations at positions 120-122 essentiallyabolished binding of 5D12, and mutations at positions 118, 119, 123 and124 substantially reduced but did not abolish binding. Thus, 5D12 bindspredominantly to positions 120-122 and, to a lesser extent, positions118, 119, 123, and 124. In each of FIGS. 6-8, 1H7 antibody is used as acontrol. 1H7 binds to residues 91-98 of alpha synuclein, and thereforeis expected to bind to the alpha synuclein regardless of presence ofmutations in residues 118-126.

The different binding specificities of 9E4 compared to 5C1 and 5D12 mayin part reflect their respective methods of production. 9E4 was made byimmunization with full-length alpha synuclein resulting in an antibodybinding a conformational epitope. 5C1 and 5D12 were made by immunizingwith a peptide of 10 amino acids resulting in a linear epitope.

FIG. 9 is a ball and stick model of the amino acids in alpha synucleinproximate to the binding sites of the 9E4, 5C1 and 5D12 antibodies. Thetwo discontinuous residues of the epitope bound by 9E4, residues 122 and125, form a pocket in the conformation of the full-length alphasynuclein protein.

Many changes and modifications can be made thereto without departingfrom the spirit or scope of the appended claims. Unless otherwiseapparent from the context, any step, feature, embodiment, or aspect canbe used in combination with any other. All publications, patent filings,web sites, accession numbers and the like mentioned in thisspecification are herein incorporated by reference in their entirety tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference. To the extent different versions of a citation exist, themost recent version at the effective filing date of the application ismeant.

1-44. (canceled)
 45. A method of treating or effecting prophylaxis of aLewy body disease comprising administering an effective regime of anantibody as defined by any of claims 1 to 36 and thereby treating oreffecting prophylaxis of the disease. 46-47. (canceled)
 48. A method ofinhibiting synuclein aggregation or reducing Lewy bodies or synucleinaggregates in a patient having or at risk of a Lewy body disease,comprising administering to the patient an effective amount of anantibody as defined by any of claims 1 to
 36. 49-56. (canceled)